A phase I/II pharmacokinetic and pharmacogenomic study of calcitriol in combination with cisplatin and docetaxel in advanced non-small-cell lung cancer.

BACKGROUND: Preclinical studies demonstrated antiproliferative synergy of 1,25-D3 (calcitriol) with cisplatin. The goals of this phase I/II study were to determine the recommended phase II dose (RP2D) of 1,25-D3 with cisplatin and docetaxel and its efficacy in metastatic non-small-cell lung cancer. METHODS: Patients were ≥18 years, PS 0-1 with normal organ function. In the phase I portion, patients received escalating doses of 1,25-D3 intravenously every 21 days prior to docetaxel 75 mg/m(2) and cisplatin 75 mg/m(2) using standard 3 + 3 design, targeting dose-limiting toxicity (DLT) rate <33 %. Dose levels of 1,25-D3 were 30, 45, 60, and 80 mcg/m(2). A two-stage design was employed for phase II portion. We correlated CYP24A1 tagSNPs with clinical outcome and 1,25-D3 pharmacokinetics (PK). RESULTS: 34 patients were enrolled. At 80 mcg/m(2), 2/4 patients had DLTs of grade 4 neutropenia. Hypercalcemia was not observed. The RP2D of 1,25-D3 was 60 mcg/m(2). Among 20 evaluable phase II patients, there were 2 confirmed, 4 unconfirmed partial responses (PR), and 9 stable disease (SD). Median time to progression was 5.8 months (95 % CI 3.4, 6.5), and median overall survival 8.7 months (95 % CI 7.6, 39.4). CYP24A1 SNP rs3787554 (C > T) correlated with disease progression (P = 0.03) and CYP24A1 SNP rs2762939 (C > G) trended toward PR/SD (P = 0.08). There was no association between 1,25-D3 PK and CYP24A1 SNPs. CONCLUSIONS: The RP2D of 1,25-D3 with docetaxel and cisplatin was 60 mcg/m(2) every 21 days. Pre-specified endpoint of 50 % confirmed RR was not met in the phase II study. Functional SNPs in CYP24A1 may inform future studies individualizing 1,25-D3.

A prospective clinical trial of cholecalciferol 2000 IU/day in colorectal cancer patients: evidence of a chemotherapy-response interaction.

BACKGROUND: We have previously reported a negative correlation between the effect of chemotherapy and 25-hydroxy vitamin D(3) (25-D(3)) levels in patients with colorectal cancer. Based on this finding, we hypothesized that the response to vitamin D(3) supplementation may be attenuated in patients with colorectal cancer. AIM: To determine 25-D(3) response to 2000 IU/day vitamin D(3) supplementation in patients with colorectal cancer. MATERIALS AND METHODS: Fifty evaluable colorectal cancer patients were treated with vitamin D(3) at 2000 IU/day for 6 months. Serum 25-D(3) levels were measured at baseline, 3, and 6 months of supplementation. RESULTS: The mean 25-D(3) level was 17.5 ng/ml at baseline, 31.6 ng/ml at 3 months, and 33.8 ng/ml at 6 months. The most important factor in determining 25-D(3) response was chemotherapy status. A rise in 25-D(3) of ≥10 ng/ml at the 3-month interval was observed in 92% of chemotherapy-free patients vs. 39% of chemotherapy patients. Similar differences in response were noted at the 6-month interval. CONCLUSION: Depressed 25-D(3) levels are common in patients with colorectal cancer. Active chemotherapy is associated with an attenuated response to 2000 IU of D(3) supplementation in this patient population. Alternative vitamin D(3) dosing schedules need further investigation in colorectal cancer patients undergoing chemotherapy.

A randomized phase II study of two doses of vorinostat in combination with 5-FU/LV in patients with refractory colorectal cancer.

BACKGROUND: Vorinostat is synergistic with 5-FU in vitro and in vivo models. A combination of these two agents was associated with clinical activity in 5-FU refractory colorectal cancer patients in a phase I clinical trial, therefore warranting the conduct of this prospective phase II study. PATIENTS AND METHODS: Patients with refractory metastatic colorectal cancer were randomized in a two-stage design to receive vorinostat at 800 or 1,400 mg/day once a day × 3, every 2 weeks. 5-FU, preceded by leucovorin, was administered as a bolus followed by a 46-h infusion on days 2 and 3 of vorinostat. A pre-specified 2-month progression-free survival (PFS) rate of 27/43 patients per arm was needed to deem an arm interesting for further investigation. RESULTS: The high-dose vorinostat arm did not reach the needed efficacy endpoint at completion of the first stage, with only 8 out of 15 patients being alive and progression free at 2 months. The low-dose vorinostat arm proceeded to accrue 43 patients with a 2-month PFS rate of 53% (23 out 43), including one partial response. The median PFS and overall survival on the low-dose arm were 2.4 and 6.5 months, respectively. Both treatment arms were well tolerated. No differences were noted in the pharmacokinetics of vorinostat at the 800- or 1,400-mg dose-levels, suggesting bioavailability saturation. CONCLUSIONS: While the addition of vorinostat to 5-FU resulted in 1 partial response and in some disease stabilizations, the limited activity does not warrant the unselected use of this combination in chemotherapy-refractory colorectal cancer.

Long-term stability of a patient-convenient 1 mg/ml suspension of tacrolimus for accurate maintenance of stable therapeutic levels.

Tacrolimus (Prograf, FK506, Fujisawa Healthcare) is a widely used immunosuppressive agent that is used both for the prevention and treatment of solid organ transplant rejection as well as for the prevention and treatment of graft-versus-host disease after allogeneic blood and marrow transplant. Oral preparations of tacrolimus are commercially available in 0.5, 1 and 5 mg gelatin capsules. Previously, only a 0.5 mg/ml oral suspension has been demonstrated to be stable for use in pediatric patients. On our bone marrow transplant service, we found that using this concentration of tacrolimus led to confusion, with patients and their caregivers confusing milligrams and milliliters, thus increasing errors with this formulation. We postulated that a 1 mg/ml oral formulation of tacrolimus would decrease the potential for medication errors. Our findings support new stability information of approximately 4 months for an extemporaneous oral suspension of tacrolimus at a concentration of 1 mg/ml.

A Phase I trial of calcitriol (1,25-dihydroxycholecalciferol) in patients with advanced malignancy.

Vitamin D is a steroid hormone best known for its activity in regulating calcium and bone metabolism. Epidemiological evidence suggests that vitamin D may play a role in inhibiting the development of colon and prostate cancer. Vitamin D receptors are expressed in many types of malignant cells; in vitro and in vivo vitamin D and vitamin D analogues are active in suppressing the development and inhibiting the growth of numerous human and animal tumors. The major toxicity of the active form of vitamin D, 1,25-dihydroxycholecalciferol (calcitriol), is the induction of hypercalcemia. There are no data indicating the maximum tolerated dose of calcitriol administered every other day (QOD) s.c. We hypothesized that this route and schedule would permit administration of higher doses of calcitriol, which might have anticancer activity. We conducted a Phase I trial of calcitriol given s.c. QOD in patients with advanced solid tumors. Thirty-six patients were entered at doses ranging from 2 to 10 microg QOD; dose-limiting toxicity (hypercalcemia) occurred in three of three patients entered at the 10-microg QOD dose. Hypercalciuria occurred at all dose levels examined. No other toxicity was seen. Assessment of serum calcitriol concentrations by a RIA revealed a decrease in concentration-time curves on day 7 compared to day 1 of therapy. A dose-dependent increase in peak serum level and estimated area under the concentration-time curve was seen. The maximum serum levels occurred at the 10-microg QOD dose: 288 +/- 74 and 321 +/- 36 pg/ml at days 1 and 7, respectively. The normal range of calcitriol serum concentration, determined using this assay, is 16-56 pg/ml. Serum calcitriol levels were maintained at near peak concentrations for at least 8 h following s.c. injection. This study indicates that substantial doses of calcitriol can be administered via this route with tolerable toxicity. Studies to explore approaches to ameliorate the hypercalcemia induced by calcitriol and to explore alternative schedules and interactions with other agents are warranted.

Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma.

All-trans retinoic acid (ATRA) inhibits human myeloma cell growth in vitro, presumably through the down-regulation of interleukin 6 receptors (IL-6R). Based on these and other studies, we initiated a phase II clinical trial using ATRA in patients with advanced refractory multiple myeloma (MM). We report that three out of six treated patients developed severe hypercalcaemia following administration of ATRA, which was accompanied by a significant rise in serum IL-6 levels. Normal calcium levels were restored after the discontinuation of the drug and the administration of standard anti-hypercalcaemic care. We suspect that down-regulation of IL-6R resulted in increased serum IL-6 levels, leading to advanced bone resorption and hypercalcaemia. We conclude that the use of ATRA in patients with advanced MM is not warranted.

Clinical pharmacology of all-trans retinoic acid.

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 are < 1 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.

Elevated plasma lipid peroxide content correlates with rapid plasma clearance of all-trans-retinoic acid in patients with advanced cancer.

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 levels > or = 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.

Clinical pharmacology of all-trans retinoic acid.

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 are < 1 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.

Modulation of all-trans retinoic acid pharmacokinetics by liarozole.

Continuous oral dosing with all-trans retinoic acid (RA) is associated with a progressive decrease in plasma drug concentrations that has been linked to relapse and retinoid resistance in patients with acute promyelocytic leukemia (APL). Since oxidation by cytochrome P-450 enzymes is critical in the catabolism of this drug, we evaluated whether pretreatment with an inhibitor of this system, liarozole, could attenuate this phenomenon. A total of 20 patients with solid tumors completed a 4-week course of all-trans RA therapy. On days 1, 2, 28, and 29, serial plasma samples were obtained from these patients after ingestion of a single oral dose (45 mg/m2) of all-trans RA. On days 2 and 29, liarozole was given 1 h prior to ingestion of all-trans RA at single doses ranging from 75 to 300 mg. The areas under the plasma RA concentration x time curves (AUCs) were then compared in the presence and absence of pretreatment. Following continuous oral treatment, the mean day-28 AUC of all-trans RA was significantly lower than the group mean level on day 1 (504 vs 132 ng h-1 ml-1; P = 0.05). This decline in plasma concentrations on day 28 was partially reversed by liarozole, which increased the mean plasma all-trans RA AUC on day 29 to 243 ng h-1 ml-1 (P = 0.004). The lowest dose of liarozole that reliably produced this effect was 300 mg. No enhanced toxicity was associated with liarozole administration. We conclude that liarozole at a dose of 300 mg effectively attenuates the induced decline in all-trans RA plasma concentrations that occurs with continuous treatment. This combination may be useful in attenuating or reversing retinoid resistance.

Constitutive variability in the pharmacokinetics of the natural retinoid, all-trans-retinoic acid, and its modulation by ketoconazole.

BACKGROUND: All-trans-retinoic acid (all-trans RA) induces complete remission in most patients with acute promyelocytic leukemia (APL). However, continuous oral dosing results in progressive decline in plasma drug concentrations, which is associated with relapse and resistance to this retinoid. We speculated that the decline in drug levels, indicating acquired resistance, resulted partly from inducible cytochrome-P450 oxidative enzymes, which can catabolize all-trans RA. PURPOSE: We studied the clinical pharmacology of all-trans RA in cancer patients to determine possible mechanisms of acquired resistance and evaluated the potential for reversal by ketoconazole, an inhibitor of cytochrome-P450 oxidative enzymes. METHODS: Serial plasma samples were obtained from 54 patients with APL or advanced lung cancer after a single oral dose of all-trans RA (45 mg/m2). In the 34 patients with advanced lung cancer, all-trans RA (45 mg/m2) was administered twice daily for 4 weeks, and, on days 2, 28, and 29, serial plasma samples were again obtained after a single 45-mg/m2 dose. One hour prior to drug administration on days 2 and 29, a single oral dose (200-1200 mg) of ketoconazole was administered. Endogenous plasma concentrations of all-trans RA and 13-cis-retinoic acid were measured in a subset of these patients and in 11 with early-stage lung cancer. RESULTS: The mean area under the curve for plasma drug concentration times time (AUC) for all-trans RA on day 1 varied substantially among patients. Compared with patients with APL, the 28 patients with advanced lung cancer who completed therapy demonstrated significantly lower AUC levels on day 1 (P = .06); a subgroup with levels less than 300 ng/mL per hour on day 1 had lower endogenous plasma all-trans RA concentrations than patients with APL or early-stage lung cancer or 14 normal subjects. Following continuous oral treatment, the mean day 28 AUC for all-trans RA was significantly lower than that on day 1 (213 ng/mL per hour versus 467 ng/mL per hour; P < .01), a decline significantly attenuated by ketoconazole, which increased the mean plasma all-trans RA AUC on day 29 to 375 ng/mL per hour (P < .01). CONCLUSION: Reported variability for the pharmacokinetics of all-trans RA may result from disease-related or population-based differences in basal catabolic rates influenced by genetic or environmental factors. However, the pattern of inducible catabolism of all-trans RA is not disease specific. Ketoconazole attenuates this accelerated catabolism, suggesting that oxidation by cytochrome-P450 enzymes is an important pathway for both constitutive and induced pathways of all-trans RA metabolism.

Specific and sensitive high-performance liquid chromatographic method with fluorescence detection for measurement of lometrexol and its polyglutamates in biologic samples.

A reversed-phase high-performance liquid chromatographic (HPLC) assay is described for the quantitative determination of lometrexol in biological samples; the assay is rapid, simple, specific, and highly sensitive. The method requires the dissociation of lometrexol from folate-binding proteins present in blood and formation of a fluorescent oxidized derivative of the compound. The dissociation of lometrexol from folate-binding proteins was achieved by acidification to pH 3.5 using ammonium formate, followed by serum protein precipitation with perchloric acid. The protein-free lometrexol was subsequently oxidized by MnO2 at 90 degrees C for 10 min. Chromatographic separation of lometrexol without interference was achieved on a C18 reversed-phase column with a convex gradient, using acetonitrile-0.1% ammonium formate, pH 7.0, as the mobile phase. In human serum and urine the calibration curve was linear between 5 and 300 nM. The lower limit of quantification was 5 nM. The method has been applied successfully to measure serum and urinary levels of lometrexol in patients.

In vitro differential metabolism of merbarone by xanthine oxidase and microsomal flavoenzymes. The role of reactive oxygen species.

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)

Lipid hydroperoxides greatly increase the rate of oxidative catabolism of all-trans-retinoic acid by human cell culture microsomes genetically enriched in specified cytochrome P-450 isoforms.

Cytochrome P-450 enzymes have been implicated in the oxidative catabolism of all-trans-retinoic acid (RA), a process that is accelerated by exposure to RA in cultured cells and rodents, and also in patients receiving RA as treatment for cancer (J.F.R. Muindi et al., Cancer Res., 52: 2138, 1992; Blood, 79: 299, 1992). Accelerated oxidation of RA could arise from an induction of RA-catabolizing P-450 isoforms or from an increase in oxidative cofactors. We have examined the efficiency of NADPH/O2 and lipid hydroperoxides (LOOH) to support oxidation of RA using human cell microsomes genetically enriched in different P-450 isoforms. The observed rate of RA oxidation using the NADPH/O2 system was slow for all isoforms (6-23 pmol/mg protein/min). LOOH-mediated oxidation was much faster (24-1078 pmol/mg protein/min), not isoform specific, but dependent upon the chemical nature of the LOOH. The order of efficiency of RA oxidation using LOOH was 13-hydroperoxy[S-(E,Z)]-9,11-octadecadienoic acid > 5-hydroperoxy[S-(E,Z,Z,Z)]-6,6,11,14-eicosatetraenoic acid > prostaglandin G2 > cumene hydroperoxide > tert-butylhydroperoxide > H2O2. Whereas submicromolar concentrations of 13-hydroperoxy[S-(E,Z)]- 9,11-octadecadienoic and 5-hydroperoxy[S-(E,Z,Z,Z)]-6,6,11,14- eicosatetraenoic acid oxidized RA at appreciable rates, micromolar concentrations were required for the other LOOH. These observations suggest that physiological LOOH, generated by the arachidonic acid-lipoxygenase system, may be involved in the self-induced oxidative catabolism of RA.

Clinical pharmacology of oral all-trans retinoic acid in patients with acute promyelocytic leukemia.

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)

Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: implications for relapse and retinoid "resistance" in patients with acute promyelocytic leukemia.

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.

Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells. Evidence that ceramide may mediate sphingosine action.

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 microM C8-cer was effective, and 3 microM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 microM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 microM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol.10(6) cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 microM) also increased cellular ceramide levels to 227 pmol.10(6) cells-1, but did not increase its own cellular level of 12 pmol.10(6) cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at effective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine may act in part by conversion to ceramide.

Clinical pharmacology of deoxyspergualin in patients with advanced cancer.

Pharmacokinetic studies were carried out in 25 patients with advanced cancer receiving deoxyspergualin (DSG), a candidate anticancer agent, in a dose-finding Phase I study. The dosage range explored was 80 to 2160 mg/m2/day for 5 days by continuous i.v. infusion. The drug levels in plasma and urine were measured by high-performance liquid chromatography with postcolumn derivatization and fluorescence detection. One drug metabolite was demonstrated in plasma and urine of treated patients. This metabolite was extracted from urine and purified to homogeneity; thereafter, it was examined by high-performance liquid chromatography, nuclear magnetic resonance, and fragmentation mass spectrometry and was demonstrated to be identical to chemically synthesized desaminopropyl-DSG. The mean steady state plasma concentrations of DSG ranged from 0.28 to 11.1 microM at, respectively, the 80- and 2160-mg/m2 dosage levels. The plasma concentration at steady state and the area under the plasma concentration versus time curve of DSG were proportional to dose (r = 0.97). Following discontinuance of the infusion, DSG was cleared from the plasma in a biexponential fashion. The mean total body clearance was 364 +/- 78 ml/min/m2. Desaminopropyl-DSG was formed extensively at all dosage levels; mean steady state plasma levels of this metabolite reached a plateau 2.65 microM at a dose of 720 mg/m2/day and did not rise with further dose increments. The urinary content of DSG was examined in 20 patients over the dosage range from 160 to 960 mg/m2/day; in this group less than 10% of the administered dose was excreted as DSG. In four patients at the 720- and 960-mg/m2/day dosage levels, the total DSG plus metabolite excretion ranged from 7 to 18% of the administered dose, with comparable quantities occurring as the parent drug and desaminopropyl-DSG.