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AP-1060 is a newly established acute promyelocytic leukemia (APL) cell line from a multiple-relapse patient clinically resistant to both all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). The line was initially derived as a granulocyte colony-stimulating factor-dependent strain that underwent replicative senescence and, following ethylnitrosourea treatment, as a phenotypically similar immortalized line. Immortalization was associated with broadened cytokine sensitivity but not growth autonomy, in contrast to three previously derived APL lines. Both the AP-1060 strain and line had shortened telomeres and low telomerase activity, while the line had higher expression of many genes associated with macromolecular synthesis. The karyotype was 46,XY,t(3;14)(p21.1;q11.2),t(15;17)(q22;q11)[100%]; the unique t(3;14) was observed in 4/9 t(15;17)-positive metaphase cells at previous relapse on ATRA therapy. The PML-RARalpha mRNA harbored a missense mutation in the RARalpha-region ligand-binding domain (Pro900Ser). This was associated with a right-shift and sharpening of the ATRA-induced maturation response compared to ATRA-sensitive NB4 cells, which corresponded to the transcriptional activation by PML-RARalphaPro900Ser of a cotransfected ATRA-targeted reporter vector in COS-1 cells. AP-1060 also manifested relative resistance to ATO-induced apoptosis at >/=1 microM, while 0.25 microM ATO stimulated limited atypical maturation. These findings suggest that AP-1060 will be useful for further assessing molecular elements involved in APL progression and drug response/resistance.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations, invariably involving the retinoic acid receptor alpha (RAR alpha) gene fused to one of several distinct loci, including the PML or PLZF genes, involved in t(15;17) or t(11;17), respectively. Patients with t(15;17) APL respond well to retinoic acid (RA) and other treatments, whereas those with t(11;17) APL do not. The PML-RAR alpha and PLZF-RAR alpha fusion oncoproteins function as aberrant transcriptional repressors, in part by recruiting nuclear receptor-transcriptional corepressors and histone deacetylases (HDACs). Transgenic mice harboring the RAR alpha fusion genes develop forms of leukemia that faithfully recapitulate both the clinical features and the response to RA observed in humans with the corresponding translocations. Here, we investigated the effects of HDAC inhibitors (HDACIs) in vitro and in these animal models. In cells from PLZF-RAR alpha/RAR alpha-PLZF transgenic mice and cells harboring t(15;17), HDACIs induced apoptosis and dramatic growth inhibition, effects that could be potentiated by RA. HDACIs also increased RA-induced differentiation. HDACIs, but not RA, induced accumulation of acetylated histones. Using microarray analysis, we identified genes induced by RA, HDACIs, or both together. In combination with RA, all HDACIs tested overcame the transcriptional repression exerted by the RAR alpha fusion oncoproteins. In vivo, HDACIs induced accumulation of acetylated histones in target organs. Strikingly, this combination of agents induced leukemia remission and prolonged survival, without apparent toxic side effects.

Arsenic trioxide (AT) has been the object of renewed interest as a therapeutic since studies in China in the late 1980s confirmed its efficacy in the treatment of acute promyelocytic leukemia (APL). These studies have been replicated in the West, with complete remissions achieved in 80% to 90% of patients with refractory or relapsed APL. The drug has been relatively well tolerated. The dose used for treatment of APL (0.15 mg/kg/d) is approximately 50% of the maximum-tolerated dose (MTD). Common side effects have included fatigue, rash, fluid retention, and QTc-interval prolongation on electrocardiogram. A "retinoic acid syndrome," similar in its manifestations to that noted after administration of all-trans retinoic acid (RA), has been observed in APL patients. Recent studies have included dose-ranging trials to determine pharmacokinetics and the optimum schedule of administration, and studies of possible mechanisms of action. Promising future trials include combining AT with RA in the treatment of newly diagnosed APL, and broadening the range of AT therapy to other leukemias, lymphomas, multiple myeloma and some solid tumors.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations always involving the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes) in t(15;17) or t(11;17), respectively. APL in patients harboring t(15;17) responds well to retinoic acid (RA) treatment and chemotherapy, whereas t(11;17) APL responds poorly to both treatments, thus defining a distinct syndrome. Here, we show that RA, As(2)O(3), and RA + As(2)O(3) prolonged survival in either leukemic PML-RARalpha transgenic mice or nude mice transplanted with PML-RARalpha leukemic cells. RA + As(2)O(3) prolonged survival compared with treatment with either drug alone. In contrast, neither in PLZF-RARalpha transgenic mice nor in nude mice transplanted with PLZF-RARalpha cells did any of the three regimens induce complete disease remission. Unexpectedly, therapeutic doses of RA and RA + As(2)O(3) can induce, both in vivo and in vitro, the degradation of either PML-RARalpha or PLZF-RARalpha proteins, suggesting that the maintenance of the leukemic phenotype depends on the continuous presence of the former, but not the latter. Our findings lead to three major conclusions with relevant therapeutic implications: (i) the X-RARalpha oncoprotein directly determines response to treatment and plays a distinct role in the maintenance of the malignant phenotype; (ii) As(2)O(3) and/or As(2)O(3) + RA combination may be beneficial for the treatment of t(15;17) APL but not for t(11;17) APL; and (iii) therapeutic strategies aimed solely at degrading the X-RARalpha oncoprotein may not be effective in t(11;17) APL.

Arsenic trioxide (As(2)O(3)) has recently been used successfully in the treatment of acute promyelocytic leukemia and has been shown to induce partial differentiation and apoptosis of leukemic cells in vitro. However, the mechanism by which As(2)O(3) exerts its antileukemic effect remains uncertain. Emerging data suggest that the endothelium and angiogenesis play a seminal role in the proliferation of liquid tumors, such as leukemia. We have shown that activated endothelial cells release cytokines that may stimulate leukemic cell growth. Leukemic cells, in turn, can release endothelial growth factors, such as vascular endothelial growth factor (VEGF). On the basis of these observations, we hypothesized that As(2)O(3) may interrupt a reciprocal loop between leukemic cells and the endothelium by direct action on both cell types. We have shown that treatment of proliferating layers of human umbilical vein endothelial cells (HUVECs) with a variety of concentrations of As(2)O(3) results in a reproducible dose- and time-dependent sequence of events marked by change to an activated morphology, up-regulation of endothelial cell adhesion markers, and apoptosis. Also, treatment with As(2)O(3) caused inhibition of VEGF production in the leukemic cell line HEL. Finally, incubation of HUVECs with As(2)O(3) prevented capillary tubule and branch formation in an in vitro endothelial cell-differentiation assay. In conclusion, we believe that As(2)O(3 )interrupts a reciprocal stimulatory loop between leukemic cells and endothelial cells by causing apoptosis of both cell types and by inhibiting leukemic cell VEGF production. (Blood. 2000;96:1525-1530)

Retinoids mediate their biological response by binding to specific nuclear receptors, including retinoic acid receptors and/or retinoid X receptors. LGD1550 is a high-affinity ligand for all three retinoic acid receptors (alpha, beta, and gamma isoforms) and a potent inhibitor of AP-1, a protein that is closely linked with trophic responses and malignant transformation. We conducted a dose ranging study to evaluate the pharmacokinetics, safety, clinical tolerance, and potential efficacy of this drug in patients with advanced cancer. Twenty-seven patients received oral doses of LGD1550 once per day at doses ranging from 20-400 microg/m2. Skin toxicity was the dose-limiting reaction at the 400 microg/m2 daily dose level. Less prominent reactions included nausea and headache. No major antitumor effects were observed. Pharmacokinetic studies in 15 patients at five dose levels showed that the peak plasma concentration (Cmax) and areas under the plasma concentration-time curve on day 1 were dose-proportional and were similar to values obtained on days 15, 29, and 84. Unlike other retinoids, LGD1550 did not induce its own metabolism, and there was little evidence of drug accumulation. The t1/2 was approximately 5 h after both the initial and repeated doses. We conclude that once-daily doses of LGD1550 of up to 300 microg/m2 are relatively well tolerated. Additional clinical explorations are warranted, especially in patients with cancers of the prostate, thyroid, head and neck, and cervix.

Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RARalpha proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid-resistant cell line derived from NB4 that no longer expresses the intact PML-RARalpha fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10(-7) to 10(-5) mol/L. As2O3 relocalized PML and PML-RARalpha onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RARalpha nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML-/- MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML-/- progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RARalpha expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL.

Gallium nitrate was originally developed as an antineoplastic agent; however, further studies have revealed that this drug has extremely potent effects on turnover of bone, and that low doses can be used to reduce bone resorption. Like the bisphosphonates, gallium nitrate has been studied in both malignant and in nonmalignant conditions. The results of randomized double blind studies have suggested that this drug has superior clinical efficacy relative to etidronate, calcitonin, and pamidronate for the acute control of cancer-related hypercalcemia. In patients with Paget's disease, low doses of gallium nitrate reduce biochemical parameters of accelerated bone turnover, including urinary excretion of calcium, hydroxyproline, and urinary collagen cross-linked N-telopeptides. Preliminary studies showed similar effects in patients with bone involvement from a wide variety of tumor types. Based on this high degree of clinical potency revealed in clinical studies, two randomized Phase III studies have been initiated in patients with bone metastases from breast carcinoma and bone involvement due to multiple myeloma. Both studies employ cyclic therapy with low dose gallium nitrate (i.e., 40 mg administered as a subcutaneous injection once daily for 2 weeks, followed by 2 weeks off treatment, recycled monthly). The endpoints of both studies are to document reductions in time to "morbid skeletal events," such as palliative skeletal radiotherapy, stabilizing orthopedic surgery, or pathologic fractures, as well as decreases in pain and analgesic requirements and improvements in mobility and other aspects of quality of life. These trials should provide definitive evidence of whether this agent is safe and effective as a treatment for bone metastases.

Inorganic arsenic trioxide (As2O3) was recently shown to induce apoptosis in NB4 promyelocytic leukemic cells. We have investigated the effects of the organic arsenical, melarsoprol (a drug used for treatment of trypanosomiasis), upon induction of apoptosis in cell lines representative of chronic B-cell lymphoproliferative disorders. An Epstein-Barr virus (EBV)-transformed B-prolymphocytic cell line (JVM-2), an EBV-transformed B-cell chronic lymphocytic leukemia (B-CLL) cell line (I83CLL), and one non-EBV-transformed B-CLL cell line (WSU-CLL) were used as targets. Dose-response experiments with melarsoprol (10(-7) to 10(-9) mol/L) were performed over 96 hours. Unexpectedly, we found that melarsoprol caused a dose- and time-dependent inhibition of survival and growth in all three cell lines. In contrast, As2O3 at similar concentrations had no effect on either viability or growth. After 24 hours, all three cell lines treated with melarsoprol (10(-7) mol/L) exhibited morphologic characteristics of apoptosis. We also observed prominent concentration-dependent downregulation of bcl-2 mRNA after 24 hours of exposure to melarsoprol in WSU-CLL, I83CLL, and JVM-2 cells. Decrease of bcl-2 protein expression was also observed in all three cell lines, whereas As2O3 had no effect on this parameter. We conclude that melarsoprol may inhibit the growth of lymphoid leukemic cell by promoting programmed cell death. Results of these studies suggest that melarsoprol shows promising therapeutic activity in these diseases, and a study to evaluate clinical effects of this drug has been initiated.

As a single agent, all-trans RA produces a higher rate of complete remission in APL than any other drug in any other neoplastic disease. The molecular findings in this illness have been exploited to develop a means of detecting and eradicating minimal residual disease. Compared with other forms of acute myeloid leukemia, this subtype is now associated with the highest proportion of patients in extended disease-free first remission who are presumably cured of their disease. APL is a prototype for understanding the pathogenesis of a malignant disease and the development of novel therapies that are targeted to specific molecular abnormalities.

The metabolic activity of cytochrome P-450 enzymes has been associated with an increased risk of developing lung cancer. We found previously that all-trans retinoic acid is catabolized by these oxidative enzymes, and that an inhibitor of this system discriminated between two populations of lung cancer patients. We examined the association between this metabolic phenotype and the risk of lung cancer in 85 subjects. The area under the plasma concentration x time curve (AUC) was calculated after a single oral dose of all-trans retinoic acid (45 mg/m2). The mean AUC for patients who had either squamous or large cell carcinomas was significantly lower than that of patients with adenocarcinomas (P = 0.0001) or control subjects (P = 0.01). Individuals with an AUC250 ng x h/ml had a greater likelihood of having squamous or large cell carcinoma (odds ratio = 5.93). This study suggests that the "rapid" catabolism of all-trans retinoic acid is linked to an increased risk of squamous or large cell cancers of the lung.

The retinoid response is mediated by families of nuclear receptors, the retinoic acid receptors (RARs), and the retinoid X receptors. All-trans retinoic acid (RA) binds only RARs and induces its own metabolism. In contrast, 9-cis RA is a newly identified agonist for both RARs and retinoid X receptors. We undertook a dose-ranging study to examine the safety, clinical tolerance, and pharmacokinetics of 9-cis RA in patients with advanced cancer. Thirty-four patients received once daily p.o. doses of 9-cis RA (administered as LGD1057) ranging from 5 to 230 mg/m2 for 4 weeks. Pharmacokinetic studies were performed on 28 patients at seven dose levels. 9-cis RA was generally well tolerated. Headache was the most common dose-limiting adverse effect. Other prominent reactions included facial flushing, myalgia, dyspnea, hypertriglyceridemia, and hypercalcemia. Relative to other retinoids, mucocutaneous reactions were mild. No major antitumor responses were observed. Pharmacokinetic analysis revealed that the day 1 area under the plasma concentration x time curves (AUCs) were proportional to the dose. Up through doses of 140 mg/m2, the day 1 AUCs were similar to those on days 15 and 29. At higher doses, however, AUCs tended to decline with repeat dosing. 9-cis RA is a novel compound that exploits a newly identified pathway of retinoid receptor biology that may be relevant to tumor cell proliferation and differentiation. We recommend a dose of 140 mg/m2 for single-agent trials utilizing a once-daily schedule of administration.

The response of acute promyelocytic leukemia (APL) peripheral blood and bone marrow cells to trans-retinoic acid (RA) was cytogenetically characterized during RA treatment using the techniques of premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH). Before treatment, the predominant immature bone marrow cells were found to have t(15;17), whereas the residual mature granulocytes were diploid and lacked evidence of the translocation. In response to RA treatment, an increase in the leukocyte count was noted. The majority of these cells exhibited a t(15;17). Subsequently (eg, between days 6 and 23), 32% to 91% of the maturing myeloid cells still exhibited t(15;17). The appearance of t(15;17) in gradually maturing elements suggests that RA contributed to a release of the maturation block of the leukemic elements. As responding patients obtained complete remission, diploid elements without evidence of the translocation prevailed in the blood and bone marrow. In 16 patients studied after 1 month in complete remission, all but 2 showed all diploid cells. The residual t(15;17) cells disappeared 18 days later in 1 patient, whereas the second patient exhibited clinical evidence of relapse 20 days later. These results suggest that response of patients with APL to RA is associated with maturation, subsequent loss of the mature leukemic elements, and preferential regeneration of normal diploid hematopoietic elements.

M195, a mouse monoclonal antibody reactive with the early myeloid antigen CD33, has been shown to target leukemia cells in patients and to reduce large leukemic burdens when labeled with 131I. A complementarity-determining region-grafted, humanized version (HuM195) has demonstrated similar targeting of leukemia cells without immunogenicity. We have studied two applications of therapy with 131I-M195. First, to intensify therapy prior to bone marrow transplantation (BMT), we combined 131I-M195 with busulfan and cyclophosphamide. Fifteen patients received first BMT for relapsed or refractory acute myelogenous leukemia or accelerated or blastic chronic myelogenous leukemia; four received second BMT for relapsed chronic or accelerated chronic myelogenous leukemia. Doses of 131I-M195 ranged from 120 to 230 mCi/m2. Few toxicities could be attributed to 131I-M195 therapy, and all patients engrafted. Eighteen patients achieved complete remission. Among those patients receiving first BMT, three have remained in unmaintained remission for 18+ to 29+ months. Six patients relapsed, including one with isolated central nervous system disease 32 months after BMT. Ten patients died in complete remission of transplant-related complications. Second, we studied whether 131I-M195 could reduce minimal residual disease and prolong remission and survival durations safely in patients with relapsed acute promyelocytic leukemia after they attained remission with all-trans-retinoic acid. Seven patients were treated with either 50 or 70 mCi/m2 131I-M195. Toxicity was limited to myelosuppression. As a measure of minimal residual disease, we monitored PML/RAR-alpha mRNA by reverse transcription PCR. Six patients had positive reverse transcription PCR assays prior to receiving 131I-M195; two converted transiently to negative. Median disease-free survival and overall survival of the seven patients were 8 (range, 3-14.5) months and 28 (range, 5.5-43+) months, respectively. This regimen compares favorably with others for relapsed acute promyelocytic leukemia. In an effort to avoid nonspecific cytotoxicity associated with 131I in future trials for minimal residual disease, we have conjugated short-range, alpha particle-emitting radioisotopes to HuM195 using a bifunctional chelate, 2-(p-isothiocyanatobenzyl)-cyclohexyldiethyl-enetriaminep entaacetic acid, with high efficiency and specific activities. 212Bi-HuM195 has demonstrated dose- and specific activity-dependent killing of HL60 cells in vitro. Injection of 213Bi-HuM195 into healthy BALB/c mice produced no effects on weight or viability.

Acute promyelocytic leukemia (APL) is characterized by a consistent chromosomal aberration that fuses the retinoic acid receptor alpha (RAR alpha) gene with the novel gene PML, resulting in the expression of a PML/RAR-alpha fusion protein. Immunohistochemical examination of APL cells shows a unique abnormal distribution of anti-PML and anti-RAR alpha antibody labeling. The PML labeling pattern observed in normal cells consists of 5 to 10 discrete spherical nuclear bodies called PODs (for "PML oncogenic domains"), whereas that of APL consists of a smaller and far more numerous speckled pattern. We examined malignant cells from patients with a variety of hematopoietic cancers by immunohistochemistry (IH) and found this abnormal PML pattern expressed in cells from patients with t(15;17)-associated leukemia but not in patients with other neoplastic disorders. IH results agreed with reverse transcription polymerase chain reaction for PML/RAR-alpha in 31 of 32 patients with acute myelogenous leukemia, including 5 of 5 patients in whom the initial clinical diagnosis of APL was not supported by cytogenetics, molecular tests, or response to all-trans retinoic acid (RA). Cells from patients with APL were examined during the course of retinoid therapy and at the time of complete remission and relapse. Reorganization of the PML labeling into PODs with normal appearance was observed in cells from patients who received RA. IH showed primarily normal PML staining during clinical remission, although the APL-specific labeling pattern was again seen in cells taken from patients at the time of relapse. Thus, IH provides an independent assay for the presence and expression of the molecular rearrangement of APL. The relative ease and speed of detecting the APL-specific PML labeling pattern should make IH a useful diagnostic tool to guide specific therapy of APL, and establish a direct assay for PML/RAR-alpha protein expression and localization in individual patient cells.

Acute promyelocytic leukemia (APL) provides a model to examine the sequential use of selective oncogene product-targeted and lineage-targeted agents. All-trans retinoic acid (RA) has been shown to produce brief remissions by a novel differentiating mechanism in most patients with APL. M195, a mouse monoclonal antibody (moAb) reactive with the cell-surface antigen CD33, can target myeloid leukemia cells in patients and reduce large leukemic burdens when labeled with 131I. We studied whether 131I-M195 could safely reduce minimal residual disease and prolong remission and survival durations in patients with relapsed APL after they had attained remission with all-trans RA. Seven patients with relapsed APL in second remission were treated with either 70 (n = 2) or 50 (n = 5) mCi/m2 of 131I-M195. As a measure of minimal residual disease, we serially monitored PML/RAR-alpha mRNA by a reverse transcription polymerase chain reaction (RT-PCR) assay. There was no immediate toxicity. Late toxicity was limited to myelosuppression, but no episodes of febrile neutropenia were seen. Six patients had detectable PML/RAR-alpha mRNA after all-trans RA therapy; two had single negative RT-PCR determinations following 131I-M195. Median disease-free survival of the seven patients was 8 months (range 3-14.5). Four patients with median follow-up of 24 months remain alive, and median overall survival exceeds 21+ months (range 5.5-33+). This regimen based on targeted therapy compares favorably to other approaches for the treatment of relapsed APL, including that used in the immediately preceding trial in which patients were induced into remission and maintained with all-trans RA, as well as other chemotherapy-based regimens. These data support further study of moAb-based therapy for minimal residual disease in acute leukemia.

All-trans retinoic acid (RA) has proven a major advance in the treatment of acute promyelocytic leukemia (APL). However, the proper management of patients who present with or develop leukocytosis during remission induction with all-trans RA is not established, nor is there a clear relation between leukocytosis and the development of the retinoic acid syndrome. We reviewed the course of our patients who underwent induction with all-trans RA to identify potential factors that might predict for the development of this syndrome and to identify which patients, if any, might specifically benefit from additional treatment with cytotoxic chemotherapy. Seventy-eight courses of all-trans RA therapy were administered to patients with a molecular diagnosis of APL. Initial and peak leukocyte counts, their rate of rise, leukocyte count criteria developed in Europe, and cell surface marker expression were all analyzed relative to subsequent development of both the RA syndrome as well as all causes of early mortality. The outcome of patients who received specific treatment for retinoid-induced leukocytosis was also examined. No factor was found to consistently predict for the development of the RA syndrome. Although the occurrence of the syndrome was positively associated with the peak value of the peripheral blood leukocyte count (P = .001), neither the initial leukocyte count nor the rate of rise in leukocyte counts on days preceding onset of the syndrome were sufficiently well-correlated to be clinically useful (P = .21). The leukocyte count criteria developed in Europe had a sensitivity of 62%, a specificity of 69%, and a positive predictive value that ranged from only 44% to 72%. However, we unexpectedly found that basal expression of CD13 (aminopeptidase N), a cell surface enzyme previously linked to tumor cell invasion and an inferior outcome in patients with acute myeloid leukemia, was highly associated with both development of the syndrome (P.05) as well as an elevated leukocyte count (P = .006). Neither low-dose chemotherapy nor leukapheresis prevented development of the syndrome nor ameliorated its effects. In fact, 9 of 11 patients who received these interventions sustained fatal or near-fatal events, most of which were due to hemorrhage. However, early treatment with a short-course of high-dose corticosteroids halted progression of the syndrome in most cases. Finally, we found that expression of the type "A" isoform of PML/RAR-alpha (also known as bcr3 or "short") was associated with a significantly shorter duration of relapse-free and overall survival (P = .005).(ABSTRACT TRUNCATED AT 400 WORDS)

Despite expectations that 2-chlorodeoxyadenosine (2-CdA) would prove active primarily in lymphoproliferative diseases, early reports suggested unexpected high activity of this drug in heavily pretreated children with acute myeloblastic leukemia (AML) at a maximally tolerated dose of 8.9 mg/m2/day for 5 days. In view of these findings, we conducted an escalating dose trial of 2-CdA in adult patients with relapsed or resistant AML. Thirty-six patients who had received extensive prior therapy were treated at 9 dose levels of 2-CdA at daily doses ranging from 5 to 21 mg/m2 for 5 days. 2-CdA eliminated leukemic blasts from the peripheral blood in 32 of 36 cases; however, bone marrow hypoplasia was seen only at daily dose levelsor = 15 mg/m2. We observed a total of 3 complete remissions: 1 at the 15 mg/m2/d dose level and 2 at the 21 mg/m2/d dose level; these responses persisted for 3, 2, and 3 months, respectively. Although prolonged myelosuppression would have been dose-limiting at 21 mg/m2/d for 5 days, the most important adverse effect was the development of a sensorimotor peripheral neuropathy. This reaction, whose onset was substantially delayed after completion of drug treatment, was observed in 2 of 5 patients at the 19 mg/m2/d level and in 4 of 4 evaluable patients at the 21 mg/m2/d level. Pathologically, this process was characterized by axonal degeneration and secondary demyelination. Other side effects included reactivation of a posttransplant Epstein-Barr virus-related lymphoma in 1 patient and tumor lysis syndrome. We conclude that the maximally tolerable dose of 2-CdA in adult patients (17 mg/m2/d for 5 days) in approximately twofold in excess of that previously reported in children and that the limiting toxic effect is a degenerative neuropathic disorder. We confirm that this drug has definite activity in AML, but the magnitude of this effect needs to be determined in larger numbers of patients who have received less extensive therapy. This agent deserves further evaluation in patients with both AML and acute lymphoblastic leukemia at these higher doses and perhaps as part of a preparative regimen for patients undergoing bone marrow transplantation.

The extramedullary collection of leukemic cells is an infrequent complication of myeloid leukemias, and extremely uncommon in acute promyelocytic leukemia (APL).The case reports of two patients with APL who relapsed with extramedullary disease and a review of the literature are presented.Two patients with cytogenetically and molecularly confirmed APL developed extramedullary relapse in skin and lymph nodes after prior treatment with all-trans retinoic acid. A review of the literature revealed only nine cases of APL complicated by extramedullary disease. However, only one of these previously reported patients was shown to have the cytogenetic abnormality characteristic of APL.To the authors' knowledge, These two patients represent the first cases of cytogenetically confirmed APL treated with all-trans retinoic acid who developed extramedullary relapse. These events, which were reported infrequently in patients treated only with cytotoxic therapy, may be more common in patients receiving all-trans retinoic acid. In view of the differentiating activity of all-trans retinoic acid, which may increase the migratory capacity of these malignant cells, it is hypothesized that this agent may predispose some patients to unusual forms of relapse.

Current data suggest that the spectacular responses seen with the single-agent use of all-trans RA in APL will probably not be replicated in other diseases in the near future. Nonetheless, that experience has revealed critical information that ultimately may be generalizable to other types of cancer. Among these lessons include the observation that in APL the t(15;17) translocation appears to dominate other cytogenetic lesions, such that therapy directed against that lesion corrects the leukemic process, irrespective of the presence of other abnormalities. In addition, the concept of induced cytodifferentiation as a practical and consistently successful method of cancer treatment has been validated. Although all-trans RA itself may not prove as useful in other diseases, it is not unreasonable to expect that other retinoids (or ligands of other nuclear receptors used alone or in combination) may be effective. Finally, although the expectation of cure in patients with advanced cancers may never be realized with these compounds, they have distinct activity as cancer chemopreventive drugs, and widespread clinical testing is ongoing. Perhaps more than any other therapy, this preventive indication affords the greatest opportunity to impact favorably on public health.

In an ongoing study, we treated 79 patients with a molecular diagnosis of acute promyelocytic leukemia (APL) using all-trans retinoic acid (RA) for remission induction. Newly diagnosed patients received cytotoxic chemotherapy for consolidation, and previously treated patients received extended all-trans RA therapy, or a radionuclide-conjugated monoclonal antibody as post-remission treatment. Unlike studies in Europe, full-dose chemotherapy was not given during induction for patients who developed leukocytosis. Overall, 43 of 49 newly diagnosed patients (88%) and 25 of 30 previously treated patients (83%) achieved complete remission. We did not encounter de novo resistance to all-trans RA in any patient who was positive for PML/RAR-alpha rearrangements by reverse transcription polymerase chain reaction analysis. Ten patients died during induction from intracranial or pulmonary hemorrhage (six patients) or the 'retinoic acid syndrome' (four patients). The use of leukapheresis or low-dose chemotherapy (hydroxyurea or cytosine arabinoside) for drug-induced leukocytosis did not decrease early mortality. Compared to historical controls, early mortality was not affected by treatment with all-trans RA; however, both relapse-free and overall survival were significantly increased. Maintenance therapy with all-trans RA was associated with short remission duration, and relapses while taking the drug were universally associated with resistance to further retinoid treatment. We conclude that the use of all-trans RA for remission induction, with or without full-dose chemotherapy, has significantly increased the survival of patients with APL. While early mortality has not yet been reduced, the avoidance of full-dose chemotherapy during induction has significantly reduced early morbidity. The major outstanding clinical issue is the development of strategies that maximize safety in high-risk patients for whom intracranial hemorrhage remains the major cause of death.

The addition of lipid hydroperoxides greatly accelerates the rate of oxidative catabolism of all-trans-retinoic acid (RA) in human cell microsomes; hydroperoxy metabolites of the arachidonate cascade are particularly active in the microsomal system. We have measured the plasma content of lipid peroxides in cancer patients during the course of therapy with RA, seeking to assess whether a correlation exists between the rate of oxidative catabolism of exogenously administered RA and whole body lipid peroxide levels. The assay used for plasma lipid peroxides is the capacity to react with thiobarbituric acid under specified conditions; the result is expressed as TBARS (thiobarbituric acid reactive substances). RA administration produced its own accelerated clearance RA within 72 h. Patients were considered to have "normal" or "rapid" baseline catabolism of RA if their Day 1 area under RA concentration over time curve was greater or less than 300 ng.h/ml, respectively. The mean plasma TBARS levels were: 12 normal volunteers = 0.14 microM; 19 "normal" RA catabolizers = 0.25 microM; and 14 "rapid" catabolizers = 0.82 microM. P = 0.008 (rapid catabolizers versus normal volunteers) and 0.05 (rapid catabolizers versus normal catabolizers). Repeat TBARS determinations were made during the course of therapy in 17 patients, all of whom converted to "rapid" RA catabolism on therapy. An increase in plasma TBARS levelsor = 20% of baseline was observed in 5 of 5 prostate cancer patients and 8 of 12 lung cancer patients treated with continuous RA therapy for 2 and 4 weeks, respectively. These observations support the hypothesis that high levels of lipid peroxides and rapid oxidative catabolism of RA are positively correlated.

The clinical pharmacology of all-trans retinoic acid (RA) has distinct differences from that of its widely studied stereoisomer 13-cis retinoic acid (cRA). RA is much more rapidly cleared from plasma following oral administration; their respective half-lives are1 h and 13 h. There is extensive accumulation of the 4-oxo-cRA in plasma following repeated doses of cRA, while 4-oxo-RA is only a minor metabolite in plasma following RA administration. The extent of isomerization in vivo differs for the two retinoids. In contrast to cRA, where up to a 1:3 ratio of RA to cRA is observed in patient plasma following drug administration, cRA concentrations in excess of 10 ng/ml are rarely observed in plasma of patients receiving exogenous RA. RA administration produces autoinduction of its own oxidative catabolism; this effect does not occur with cRA. These pharmacokinetic differences have been observed in leukemia and solid tumor patients. Detailed analysis of the results of the population studied suggest that both constitutive and RA-induced hypercatabolism of RA occurs. Both of these hypercatabolic states can be modulated by concurrent administration of ketoconazole, an inhibitor of cytochrome P-450 and lipoxygenase-mediated oxidations.

Acute promyelocytic leukemia (APL) is characterized cytogenetically by a balanced reciprocal chromosomal translocation t(15;17) (q22;q21). This translocation involves the retinoic acid receptor-alpha (RAR-alpha) on chromosome 17 and the promyelocytic leukemia locus (PML) on chromosome 15 and results in the transcription of novel fusion messenger RNAs. In this study, pulsed-field gel electrophoresis (PFGE) was applied to the detection of the t(15;17) translocation in twenty-six clinical specimens cytologically diagnosed by French-American-British criteria as APL. This technique could readily be applied to both fresh and nonviably frozen tumor samples. In 24 of 26 samples, rearrangements of the PML and RAR-alpha, loci could be detected by Southern blotting after digestion with MluI and BssHII. Furthermore, co-migration of the rearranged fragments, detected by hybridization to probes for the PML and RAR-alpha genes, demonstrated that these loci were juxtaposed. The translocation was detected in specimens at the time of initial diagnosis, on differentiation therapy with retinoic acid and at the time of relapse. The diagnostic accuracy was compared to cytogenetics and the reverse transcriptase-polymerase chain reaction for the novel PML-RAR-alpha fusion transcript. The samples from two patients were negative by all three diagnostic methods, and both of these patients failed to respond to all-trans retinoic acid. In the other 24 APL samples, cytogenetics was positive in only 76.9% of the cases, whereas both reverse transcriptase-polymerase chain reaction and PFGE methods detected the translocation in 100% of the cases. Thus, PFGE can readily detect the t(15;17) translocation in both viable and nonviable clinical specimens and can improve the diagnostic accuracy of morphology and cytogenetics in APL. In contrast to conventional electrophoresis based on rearrangement of RAR-alpha, the ability to demonstrate directly co-migration of the PML and RAR-alpha loci enables this method to distinguish the t(15;17) translocation from variant translocations such as the t(11;15). Because PFGE can be performed on nonviable, frozen tumor samples, it could be diagnostically useful in APL when the RNA-based reverse transcriptase-polymerase chain reaction cannot be performed.

The characteristic reciprocal translocation t(15;17) of acute promyelocytic leukemia (APL) disrupts the PML gene on chromosome 15 and the retinoic acid receptor-alpha (RAR-alpha) gene on chromosome 17. PML/RAR-alpha fusion mRNAs are then transcribed and can be detected by a newly described reverse transcription polymerase chain reaction (RT-PCR) assay. Using RT followed by nested PCR amplification for PML/RAR-alpha, we serially evaluated bone marrow aspirates from patients with APL who were treated with all-trans retinoic acid (RA) for induction, followed by all-trans RA as maintenance or cytotoxic drugs as consolidation. At diagnosis, PML/RAR-alpha mRNA was detected in all patients. After initial therapy with all-trans RA, the RT-PCR assay remained positive after induction of complete remission in 31 of 32 evaluable patients. Maintenance treatment by all-trans RA alone was associated with persistent assay positivity and subsequent clinical relapse in 13 of 13 patients. By contrast, the test became negative in 19 of 20 newly diagnosed patients who received consolidation chemotherapy; the 1 patient who remained positive relapsed at 12 months. Three of the 19 assay-negative patients later converted to positive and subsequently relapsed; the remaining 16 patients have remained RT-PCR negative in sustained first remission, with a median follow-up duration that exceeds 24 months (range, 12+ to 34+ months). Despite induction of complete remission in a high proportion of patients, all-trans RA rarely eradicates molecular evidence of disease in patients with APL; however, subsequent treatment with cytotoxic chemotherapy frequently converts the RT-PCR assay for PML/RAR-alpha to negative. Serial negative tests are associated with prolonged disease-free survival, whereas persistence of a positive test after treatment is highly correlated with subsequent relapse. This test identifies patients in remission at high risk for relapse who may benefit from additional antileukemic therapy.

PURPOSE A randomized, double-blind, dose-ranging study of single-dose intravenous (IV) therapy with alendronate sodium (aminohydroxybutylidene bisphosphonate) was performed in patients with cancer-associated hypercalcemia. PATIENTS AND METHODS Patients with hypercalcemia who had not received antitumor therapy in the preceding 7 days were treated with 48 hours of IV hydration. Patients with persistent hypercalcemia (albumin-corrected serum calcium concentration [CSCC] > or = 11.5 mg/dL) were randomly assigned to receive 2.5, 5, 10, or 15 mg of alendronate infused over 2 hours, or 10 mg of alendronate infused over 24 hours. Fifty-nine patients were treated and 50 patients were assessable for the dose-response relationship. RESULTS Normalization of CSCC (< or = 10.5 mg/dL) was achieved in 22%, 82%, 75%, and 90% of assessable patients in the 2.5-, 5-, 10- (2- and 24-hour groups pooled), and 15-mg dose groups, respectively, within 8 days of therapy. Doses > or = 5 mg were significantly superior to the 2.5-mg dose level (P < .05). There was no significant difference in the minimum CSCC achieved between the 2- and 24-hour infusions of the 10-mg dose. Based on an intent-to-treat analysis of all randomized patients, the overall complete response rate was 74% for dose levels greater than 2.5 mg. For assessable patients who responded to > or = 5 mg of alendronate, the estimated median duration of normocalcemia was 10 days (range, 1 to 25). The estimated median time to relapse (CSCC > 11.5 mg/dL) was 15 days from initial treatment and 12 days from initial response, respectively. Adverse events included a transient febrile response in 34% of patients and eight episodes of reversible elevations in serum transaminase levels among treated patients. CONCLUSION While a statistically significant dose-response relationship was not clearly evident at doses greater than 5 mg, single doses of > or = 5 mg alendronate sodium effectively lowered serum calcium concentrations and were well tolerated in the treatment of cancer-associated hypercalcemia.

Merbarone (MB), a nonsedating derivative of thiobarbituric acid, was recently found to induce profound hypouricemia. When incubated with xanthine oxidase (XO) and hypoxanthine in vitro, MB is both an inhibitor of XO and degraded by the XO-hypoxanthine interaction. Compared with allopurinol (Ki = 0.025 microM), MB is a very weak inhibitor of XO (Ki = 51 +/- 8 microM). MB interacts with XO in the presence of hypoxanthine to yield three chromatographically separate products. One of these products has been identified by HPLC retention time and spectral characteristics as 2-oxo-2-desthiomerbarone (2-oxo-MB). The other two products are thought to be S-oxide intermediates in the oxidative desulfuration of this drug. Formation of these products was blocked by catalase, suggesting that the conversion was dependent on reactive oxygen species (especially H2O2) generated by the hypoxanthine-XO system. This suggestion was confirmed by incubating MB with H2O2. In vitro studies with rat liver microsomes have documented the formation of 2-oxo-MB and 4'-OH-MB (4'-OH-MB), the latter being identified by the characteristic HPLC retention time of its acetylated derivative. The formation of 4'-OH-MB has many characteristics of a cytochrome P-450-dependent monooxygenase reaction (NADPH requirement and SKF 525-A inhibition); formation of 2-oxo-MB occurs by a different mechanism that is, as yet, uncharacterized. Incubation of kidney microsomes with MB generated 2-oxo-desthiomerbarone but no detectable 4'-OH-MB.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloroquinoxaline sulfonamide (CQS) is a halogenated heterocyclic sulfanilamide identified by the in vitro human tumor colony-forming assay as an active agent in a variety of human solid tumors. In this phase I study, 182 courses of CQS were administered intravenously every 28 days to 88 patients at doses ranging from 18 to 4870 mg/m2. Hypoglycemia associated with hyperinsulinemia was the dose-limiting adverse effect at 4870 mg/m2. Supraventricular tachyarrhythmias were observed at doses4000 mg/m2. Less common reactions included infusion site phlebitis, nausea, anemia, alopecia, perioral numbness, and diarrhea. Cumulative toxicity was not observed. Minor objective antitumor responses were noted in 7 patients; 6 of the 7 responses occurred in patients with non-small cell lung cancer. Results of pharmacokinetic studies were consistent with the preclinical observations that CQS is highly bound to plasma protein. Plasma elimination followed a two-compartment model; the mean t 1/2 alpha was 2.7 +/- 0.3 h and the t 1/2 beta was 52 +/- 6 h (+/- SE). The total body clearance and the volume of distribution at steady state of CQS both increased with the dose (distribution at steady state, 3.7-10.5 liter/m2; total body clearance, 53-264 ml/h/m2 for doses of 18-4060 mg/m2) and may reflect saturation of the protein binding and "free" drug clearance. Although inactive against common animal tumors in preclinical screening systems both in vitro and in vivo, CQS has demonstrated definite activity in the human tumor stem cell colony-forming assays, as well as modest anticancer activity in this phase I study in patients with advanced solid tumors. The pharmacokinetic results and the limiting effect of transient hypoglycemia suggest that considerably higher cumulative doses of CQS could be administered using a more frequent dosing schedule.

Important new information has been gained concerning the etiology of cancer-related hypercalcemia and thus treatment recommendations are changing. The most common cause of hypercalcemia in cancer patients is the parathyroid hormone-like peptide, PTH-RP. Patients with elevated serum calcium due to elaboration of parathyroid hormone-like peptide commonly present with hypophosphatemia and a relatively resistant form of hypercalcemia. Our new knowledge has led to recommendations against massive amounts of IV fluids and large doses of diuretics, which restore normocalcemia in only a minority of patients. New potent drugs such as pamidronate and gallium nitrate directly inhibit accelerated bone resorption. Thus, consideration should be given to the early administration of antiresorptive drugs immediately after intravascular volume has been repleted and urinary output has been established.

All-trans retinoic acid (RA) induces leukemic cell differentiation and complete remission in a high proportion of patients with acute promyelocytic leukemia (APL). However, remissions induced by all-trans RA tend to be brief, and relapses are associated with resistance to further treatment in vivo, although the leukemic cells appear to retain sensitivity to the cytodifferentiating effects of all-trans RA in vitro. The clinical pharmacology of all-trans RA was examined in 13 patients with APL. The drug was administered at a constant dose of 45 mg/m2/day, given as a single dose on the first day of therapy and in two divided doses thereafter. Plasma and urinary concentrations of the parent drug and metabolites were quantitated by reverse-phase high-performance liquid chromatography and, where required, by a combination of normal-phase liquid chromatography/negative chemical ionization mass spectrometry. In patients with APL, basal levels of endogenous retinol and natural retinoids were within the normal range. Peak plasma levels of all-trans RA (347 +/- 266 ng/ml, mean +/- SD) were reached 1-2 h after drug ingestion and decayed in a monoexponential fashion with a half-life of 0.8 +/- 0.1 h. The only drug metabolite detected in plasma or urine was 4-oxo-all-trans RA (present in urine as the glucuronide conjugate). This metabolite accounted for less than 10% of the circulating drug in plasma, and its cumulative urinary excretion accounted for less than 1% of the administered dose. The drug was not found in cerebrospinal fluid. Continued oral administration of all-trans RA was associated with a significant decrease in both the plasma peak levels and the area under the concentration-time curve (P = 0.01 and 0.004, respectively) when measured after 2-6 weeks of treatment. We previously reported that a decrease in plasma area under the concentration-time curve was highly correlated with clinical relapse. Observations in a subset of patients in this study suggested that, in fact, the major decrease occurred early, within the first 7 days of treatment. These changes were associated with a 10-fold increase in urinary excretion of 4-oxo-all-trans RA glucuronide, suggesting that the accelerated clearance from plasma was associated with increased drug catabolism. The rapid disappearance may explain early relapse from remissions induced by all-trans RA; clinical "resistance" to all-trans RA may either wholly or in part result from an inability to sustain effective plasma concentrations of all-trans RA during continuous treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Although all-trans retinoic acid (RA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), all groups have described clinical relapses despite continued RA treatment. This finding suggests that resistance to the cytodifferentiating effects of the retinoid had been acquired. To investigate potential mechanisms of clinical resistance to RA, we serially evaluated the clinical pharmacology of the drug in APL patients treated with this agent. Leukemic cells from patients relapsing from RA treatment were cultured in the presence of RA and examined for evidence of morphologic maturation. We also studied messenger RNA expression of the newly described gene product of the (15;17) translocation in APL, PML/RA receptor-alpha (PML/RAR-alpha). Serial pharmacokinetic studies showed that continuous daily RA treatment was associated with a marked decrease in plasma drug concentrations at the time of relapse compared with the initial day of therapy. Doubling the RA dose in six patients failed to reinduce response at the time of relapse and also failed to significantly augment plasma RA concentrations. However, leukemic cells obtained at the time of relapse from four patients retained in vitro sensitivity to the differentiating activity of RA (10(-6) mol/L). No change was observed in the pattern of PML/RAR-alpha expression assessed by Northern blot analysis at the time of relapse compared with pretreatment in two patients who were tested. These results indicate that clinical relapse and "resistance" to continuous treatment with all-trans RA in APL is associated with progressive reduction of plasma concentrations, potentially to levels below those that sustain differentiation of leukemic cells in vivo. Long-term success of this treatment will require the development of strategies that circumvent this pharmacologic phenomenon.

2510 Background: As a class, taxanes have wide interpatient (pt) pharmacokinetic (PK) variability at their effective dose, which has been associated with adverse reactions, especially neutropenia. Tesetaxel is an oral taxane with Phase 2 activity in pretreated pts with colorectal, non-small cell lung, gastric, and breast cancers. Tesetaxel is not a substrate for P-glycoprotein; it is associated with lower neurotoxicity at equi-myelotoxic does, and it displays a long terminal half-life in plasma (∼180 hrs). We explored PK and clinical safety to determine the feasibility of using flat dosing in future studies. Methods: PK parameters were evaluated in 21 pts treated at the maximum tolerated dose (27 mg/m2) either alone or in combination with capecitabine. We then compared actual dose (total mg) with the weight-based dose and correlated the incidence and severity of Grade 3–4 neutropenia with weight-based vs. flat dosing. Results: A total of 174 pts were treated with tesetaxel in Phase 2 trials at doses of 27 or 35 mg/m2 once every 3 weeks. For pts prescribed with weight-based dosing (27 mg/m2), mean Cmax in 3 separate cohorts was 24.6, 42.5, and 47.9 μg/L, with AUC0-inf of 1363, 1592, and 1663 h*μg/L, respectively. In 103 pts prescribed a dose of 27 mg/m2, 17% and 16% of pts received doses of 40 mg or 60 mg, respectively, whereas 67% received 50 mg. Correlations between drug exposure AUC0-inf and actual dose (mg and mg/m2) across the entire range of administered doses showed r2 values of 0.455 and 0.492, respectively. Similar correlations after a second repeat dose showed r2 values of 0.731 and 0.653, respectively. The incidence of Grade 3–4 neutropenia at 27 mg/m2 was 33%. Conclusions: PK and clinical safety suggest that weight-based dosing offers no substantial advantage compared with flat dosing. Future studies could incorporate a starting dose of 50 mg administered once every 3 weeks. Flat dosing should also be tested using different schedules, including weekly x 3 weeks and “metronomic dosing” (1–2 mg/d). [Table: see text]

Neuropathy and myopathy are common sequelae of intensive chemotherapy protocols that contain vincristine and corticosteroids. The authors prospectively monitored the evolution of neuropathy and myopathy during an intensive 12-week chemotherapy program for patients with intermediate and high-grade non-Hodgkin's lymphoma. In this study, vincristine was administered by bolus injection followed by a 3-day continuous intravenous (IV) infusion (total dose of 2.0 mg/m2 every other week); the maximum dose of vincristine was not arbitrarily limited. Cronassial, a mixture of four naturally occurring gangliosides, was administered in a randomized double-blind test to evaluate whether this agent could prevent vincristine-induced neuropathy. High doses of dexamethasone (50 mg/d for 3 days weekly or every other week) were also prescribed. Patients were monitored every 4 weeks with comprehensive physical and neurologic examinations and electrophysiologic studies of peripheral nerve function. Twenty-seven patients were fully evaluable. Weakness was a prominent adverse reaction in this study, and all patients had moderate to severe signs and symptoms of neuropathy and myopathy. Cronassial (100 mg) administered by intramuscular (IM) injection daily provided no protection against the development of neuropathic symptoms. Vincristine typically impaired fine-motor coordination initially, whereas corticosteroids were associated with delayed development of proximal muscle weakness. Results of electrodiagnostic studies did not add to the clinical examination results. The authors conclude that symptomatic weakness due to neuropathy or myopathy appears in a predictable manner during intensive vincristine/corticosteroid-based treatment protocols. Simple clinical tests can be used to rapidly distinguish between toxic effects due either to vincristine or corticosteroids, and routine implementation of these tests can prevent inappropriate dose attenuation of these agents.

Merbarone, a nonsedating derivative of thiobarbituric acid, has demonstrated excellent activity against certain murine tumors, including L1210 and P388 leukemias, B16 melanoma, and M5076 sarcoma. Preclinical studies suggested that the antitumor effects of this drug were schedule dependent, since repeated dosing increased killing of tumor cells when compared to intermittent injections. We have completed a Phase I clinical and pharmacological study of merbarone in which the drug was administered both as a 2-h infusion and as a continuous i.v. infusion over 24 h. In view of the increased toxicity observed in animals following bolus injections and the possibility of schedule-dependent anticancer activity, a schedule of drug administration daily for 5 days was selected. Fifty patients with advanced cancer were treated at dose levels that ranged from 100 to 1500 mg/m2/day. When the drug was administered by peripheral vein, phlebitis was observed at the infusion site at daily doses greater than or equal to 150 mg/m2. Therefore, all patients who received drug doses greater than or equal to 200 mg/m2 were treated by continuous i.v. infusion using central venous catheters. Renal insufficiency, initially observed at a dose of 1000 mg/m2/day, was the dose-limiting toxic reaction at 1500 mg/m2/day. Three of five patients treated at the highest dose level were unable to complete the infusion due to this effect. Marked hypouricemia was observed in all patients. Other toxic effects were mild and included nausea, fatigue, leukopenia, thrombocytopenia, and anorexia. Alopecia was noted in several patients who received doses greater than or equal to 1000 mg/m2/day. No major antitumor effects were observed. Dose-dependent, steady-state plasma concentrations of merbarone were reached within 24-48 h after beginning the continuous i.v. infusion. Elimination of drug from plasma followed a two-compartment model, with a t1/2 alpha of 4.2 h and a t1/2 beta of 15.3 h. Renal excretion of merbarone and its major metabolites accounted for less than 30% of the administered dose. We conclude that merbarone is relatively well tolerated with few constitutional symptoms. The current formulation of the drug causes phlebitis when administered by peripheral vein, and renal insufficiency is commonly observed at daily doses which exceed 1250 mg/m2. The recommended dose for extended Phase II evaluation is 1000 mg/m2/day daily for 5 days administered by central venous catheter.

Following in vivo administration of gallium nitrate, the greatest concentrations of the therapeutic element gallium localized in the metaphysis and at the endosteal and periosteal surfaces of the diaphysis. These are the regions of greatest metabolic activity, where new bone formation and remodeling are occurring. The lowest levels of gallium were noted in the mid-cortical region of the diaphyseal shaft where bone turnover is least. The accumulation of gallium in the metaphysis was associated with a concomitant fall in iron and zinc. The gallium-induced change in the metaphysis may reflect a subtle modulation of metal dependent enzymes that are necessary for the active bone modeling that occurs in this bone region. X-ray microscopy has provided the first insights into the localization and possible mechanisms of action of gallium in bone.

We treated 51 patients with advanced malignant lymphoma refractory to conventional therapy with methyl-glyoxal-bis(guanylhydrazone) (methyl- GAG) at doses ranging from 400 to 800 mg/sq m. Therapy was started on a weekly schedule and was switched to every other week in responding patients at the onset of toxicity. Partial responses were observed in 6 of 13 evaluable patients with Hodgkin's disease (46%), 5 of 10 patients with diffuse poorly differentiated lymphocytic lymphoma (50%), 2 of 4 patients with nodular poorly differentiated lymphocytic lymphoma (50%), and 3 of 13 patients with diffuse histiocytic lymphoma (23%). Two of six patients with mycosis fungoides showed objective improvement in cutaneous disease. Toxicity was generally mild and included muscular weakness, myalgia, mucositis, and diarrhea; two patients developed bronchospasm following drug infusions. We conclude that methyl-GAG has major antitumor activity when administered on this schedule to patients with advanced malignant lymphoma. The low degree of toxicity, unique mechanism of action, and minimal myelosuppressive effects suggest that methyl-GAG will prove useful in future trials of combination chemotherapy regimens for the treatment of lymphoma.

Aclacinomycin A (aclarubicin; ACM) is a new class II anthracycline antibiotic. Preclinical studies suggested that ACM had approximately equivalent antitumour activity but produced substantially less cardiotoxicity compared to other anthracyclines. Because of the recognized importance of these compounds in the treatment of haematological tumours, clinical trials of ACM were initiated in the late 1970s. ACM has been extensively evaluated in patients with relapsed leukaemia and advanced malignant lymphoma. Analysis of results compiled from Europe, Japan, and the United States shows that ACM is probably equivalent to doxorubicin for remission induction of patients with relapsed acute non-lymphoblastic leukaemia. Initial studies using ACM alone and in combination with standard cytotoxic drugs in previously untreated patients compare favourably with the best standard treatment for this disease. The antitumour activity of ACM in patients with acute lymphoblastic leukaemia or malignant lymphoma who have previously received doxorubicin or daunorubicin is low, and the issue of whether ACM lacks clinical cross-resistance to other anthracyclines is unresolved. Acute cardiac arrhythmias have been observed following administration of ACM, but congestive cardiomyopathy has been uncommon. Results to date all indicate that ACM has fulfilled its early expectations of antileukaemic activity and reduced toxicity. These hypotheses should now be evaluated in prospective, randomized trials with conventional anthracyclines.

Previous studies regarding sites of platelet destruction in patients with the Kasabach-Merritt syndrome are conflicting. The authors recently studied an adult patient with multiple large hemangiomata, thrombocytopenia, and intravascular coagulation by external imaging following the injection of autologous Indium-111 labeled platelets. Sequential images showed prompt accumulation of platelet-associated radioactivity in areas within the right hemithorax which corresponded to certain tumors noted on the chest roentgenogram. Despite the presence of multiple other lesions in bone and soft tissues, platelet radioactivity was otherwise normally confined to liver and spleen. Using data obtained from serial images, it was shown that radioactivity within the thoracic masses actually increased over time. These data indicate that platelet consumption occurred as an active process and that localization was not a result of tumor vascularity. It is concluded that platelets are locally consumed within certain hemangiomata. However, within the same individual, there may exist considerable heterogeneity among these tumors with respect to platelet-trapping ability. In similar patients with multiple tumors, indium-platelet scanning might be used to direct local therapy to particular lesions in an effort to correct the thrombocytopenia.

We treated 13 adult patients with acute leukemia or chronic myelocytic leukemia (CML) in blast phase using succinylated Acinetobacter glutaminase-asparaginase (SAGA) administered on a daily dose schedule. SAGA reduced the peripheral blast count in two patients with acute lymphoblastic leukemia and two with blastic CML; however, no patient achieved either complete or partial remission. Marked central nervous system toxic effects (encephalopathy and coma) were observed, limiting treatment in patients whose disease appeared responsive; this effect finally prompted early discontinuance of the trial. Other toxic effects observed included nausea, hyperglycemia, and respiratory alkalosis. Hypersensitivity reactions to the enzyme were not seen. Pharmacologic analyses showed that prolonged blood glutamine depletion was achieved only by daily enzyme administration; however, we noted the importance of performing amino acid analysis on blood which was deproteinized immediately following phlebotomy. Our results demonstrate excessive central nervous system toxicity when glutaminase-asparaginase is administered on a daily schedule. Because of this effect, we propose that future trials of similar enzymes be limited to short courses of enzyme therapy, possibly with the addition of antimetabolites or amino acid analogs, which could enhance the antitumor effect without increasing toxicity.

Enzymuria and beta 2-microglobulinuria are sensitive markers of injury to renal tubular cells. We evaluated these markers in 41 patients with advanced malignant lymphoma who received gallium nitrate administered as a continuous infusion during a recent phase I-II study. In contrast to our findings with cisplatin, we observed no significant increase in enzyme excretion or beta 2-microglobulinuria in these patients even after long-term treatment. Our results indicate that gallium nitrate administered by infusion does not produce cumulative renal tubular toxicity.

Current treatment of cancer-related hypercalcemia is limited by agents of limited effectiveness or excessive toxicity. Gallium nitrate is a new drug which both inhibits bone resorption and increases calcium content of bone. We have now treated 39 episodes of hypercalcemia with gallium nitrate administered as a continuous i.v. infusion for 5-7 days at 3 daily dose levels (100 and 200 mg/m2, and 50 mg/m2 by brief infusion followed by 150 mg/m2). Nadir calcium values were significantly lower (9.2 +/- 1.5 mg/dl) for patients who received the highest dose relative to patients who received the lowest dose (10.5 +/- 1.6 mg/dl, P less than 0.001). While the actual percentage of patients who achieved normocalcemia was higher at the highest dose relative to the lowest dose (86 versus 60%), this difference was not statistically significant. Mean serum concentration of inorganic phosphorous declined significantly for all patients from 2.9 +/- 0.86 mg/dl at base line to 1.8 +/- 0.66 mg/dl (P less than 0.001). Pharmacokinetic studies suggested that a threshold plasma gallium concentration of approximately 1 microgram/ml must be attained to achieve acute normalization of elevated serum calcium levels. Steady-state plasma gallium levels were attained after 48 h; there was no evidence of drug accumulation in plasma after 2 days. Effects on serum creatinine concentration were negligible, and there were no other toxic reactions. These data confirm preclinical experiments which suggested that inhibition of bone resorption by gallium nitrate is dependent upon the dose and duration of drug exposure. We conclude that gallium nitrate is effective treatment for cancer-related hypercalcemia. The drug is now being evaluated against standard treatment in a randomized, double-blind trial.