537.Full.Pdf

Vol. 3, 537-543, April 1997 Clinical Cancer Research 537

A Pilot Clinical/Pharmacological Study of the Protein Kinase C-specific Inhibitor Safingol Alone and in Combination with Doxorubicin

Gary K. Schwartz,1 David Ward, Leonard Saltz, INTRODUCTION Ephraim S. Casper, Tara Spiess, Eillen Mullen, PKC2 is a phospholipid-dependent serine/threonine kinase that plays a pivotal role in many of the signal transduction events Jim Woodworth, Robert Venuti, Peter Zervos, related to tumorigenesis. Activation of cell surface receptors by Anne-Mane Storniolo, and David P. Kelsen extracellular molecules such as growth factors and hormones can Department of Medicine, Division of Solid Tumor Oncology, stimulate phosphatidylinositol turnover, leading to elevated levels Gastrointestinal Oncology Section, Memorial Sloan-Kettering Cancer of inositol triphosphate and sn-l,2-diacylglycerol, the endogenous Center, New York, New York 10021 [G. K. S., L. S., E. S. C., T. S., E. M., D. P. K.], and Eli Lilly Research Lab, Indianapolis, Indiana ligand that activates PKC (1 , 2). These extracellular signaling 46285 [D. W., J. W., R. V., P. Z., A-M. S.] molecules, as well as their receptors, are altered in their effect by cellular oncogenes, and diacylglycerol is known to be elevated in cells transformed by ras, sis, and src (3). In addition, activation of ABSTRACT PKC leads to the expression of nuclear proto-oncogenes (e.g., myc, We performed a pilot clinical trial with safingol (L- fos, cis, and fins) associated with cellular proliferation. PKC is threo-dihydrosphingosine), a protein kinase C-specific in- activated by phorbol esters, such as phorbol myristate acetate, hibitor that potentiates the effect of doxorubicin (DOX) in which are potent tumor promoters in mouse skin (4). Increased tumor-bearing animals. Safingol was initially administered tumorigenicity also correlates with overexpression of certain PKC as a 1-h infusion at escalating doses. Fourteen days later, isoforms in Nil-I 3T3 cells inoculated into nude mice (5). The patients received the same dose of safingol in combination involvement of PKC in so many aspects of aberrant growth regu- with a fixed dose of DOX. The combination was repeated at lation suggests that the modulation of PKC activity might be an 3-week intervals. Safingol dose levels ranged from 15 to 120 effective target for anticancer therapy. mg/m2. The plasma levels achieved at the final dose level Safingol (Fig. 1), the L-threo enantiomer of dihydrosphin- were comparable to those associated with potentiation of gosine, is a PKC-specific inhibitor that inhibits PKC enzyme ac- DOX in animals. The mean Cmax and area under the curve tivity in micromolar concentrations (6). In vitro studies of safingol for safingol at the 120 mg/m2 dose level were 1040 ± 196 have demonstrated reversal of multidrug resistance in DOX-resist- ng/ml and 1251 ± 317 mg x h/mI, respectively. The mean ant cell lines (7, 8). Safmgol has also been shown to increase the plasma half-life for safmgol was 3.97 ± 2.51 h, the mean activity of DOX and other chemotherapeutic agents, including estimated clearance was 3140 ± 765 mI/mm, and the mean mitomycin C, by enhancing chemotherapy-induced apoptosis, even volume of distribution was of 995 ± 421 liters. Coadminis- in tumor cell lines that were resistant to chemotherapy by virtue of tration of a fixed dose of DOX did not significantly change a mutation in p53 (6). In vivo, as a single agent, safingol has shown the pharmacokinetics of safingol, nor did increasing doses of little direct antitumor activity. However, in a series of murine tumor safingol significantly affect the pharmacokinetics of DOX. models and human tumor xenografts, safingol has been shown to Minor responses were observed in three patients with pan- significantly modulate the antitumor effect of DOX and cisplatin creatic cancer and one patient with angiosarcoma of the (9, 10). This was achieved without an increase in chemotherapy- induced bone marrow suppression or major organ toxicity (i.e., scalp. This pilot Phase I study indicates that the protein ear or kidney). In these studies, nontoxic serum levels of safingol could kinase C inhibitor safingol can be given safely with 45 be attained at concentrations associated with inhibition of PKC mg/rn2 of DOX at a dose that is potentially pharmacologi- enzyme activity (1 1). cally active without dose-limiting toxicity. Because the in vivo activity of safingol appeared to be greatest when combined with chemotherapy and because this was associated with established serum safingol levels, we performed a pilot clinical study of safingol and DOX with pharmacological end points designed to determine whether we could

Received 6/4/96; revised 12/18/96; accepted 12/30/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to 2 The abbreviations used are: PKC, protein kinase C; DOX, doxorubi- indicate this fact. I To whom requests for reprints should be addressed, at Department of cm; PT, prothrombin; P’FT, partial thromboplastin; CBC, complete Medicine, Division of Solid Tumor Oncology, Gastrointestinal Oncol- blood count; DLT, dose-limiting toxicity; HPLC, high-performance ogy Section, Memorial Sloan-Kettering Cancer Center, New York, NY liquid chromatography; AUC, area under the curve; HNTD, highest 10021. Phone: (212) 639-8324; Fax: (212) 717-3320. nontoxic dose.

QH CH3(CH2)1 4AyOH U U-

NH2 Day# 1 14 35

Fig. 1 Safingol (L-threo-dihydrosphingosine). I Safingol (infusion over I hour) Doxorubicin (bolus)

achieve nontoxic levels of safingol in serum associated with Fig. 2 Treatment scheme of Phase I study for safingol/DOX. Patients chemopotentiation of DOX in animals. were treated in cohorts of three to six patients with safingol on day 1. If there was no acute toxicity, they were again treated with the same dose of safingol on day 14, followed in 1 h by a fixed dose of DOX. If there PATIENTS AND METHODS was no dose-limiting toxicity, they could then be retreated with the same Patients. Patients treated as part of this clinical trial had to combination at the same doses 21 days later (day 35). Subsequent meet the following criteria. All patients had to be 18 years of age cohorts were treated with an increased dose of safingol according to the same treatment scheme and with the DOX dose fixed at all safingol dose with histologically confirmed carcinoma by the Pathology Depart. levels. ment of Memorial Hospital. Patients had to have cancer refractory to standard therapy (or for which there was no standard therapy) and not curable by surgery or radiation therapy. A Karnofsky performance status 60% with a life expectancy of at least 12 measurement of all measurable disease, as well as signs and weeks was required. Previously treated patients were accepted. symptoms. Pretreatment laboratory studies included a CBC, They may not, however, have received myelosuppressive chemo- platelet count, differential, biochemical screening profile, serum therapy or radiation therapy to major bone marrow-containing creatinine, PT, P’fl’, chest X-ray, and urinalysis, and serum areas within the previous 4 weeks (6 weeks for prior nitrosurea or pregnancy test for females. Radiological studies with measure- mitomycin C), and they must have recovered from the marrow ment of the tumor indicator lesion(s) were performed as clini- toxic effects of prior chemotherapy and radiation. cally indicated. Pretreatment gated pool heart scan at rest and a All patients must have had a WBC count 4000/p.l, a total 12-lead EKG were required. neutrophil count 1500/p.l, a platelet count l50,000/p.l, and a Safingol Administration. Safingol is the non-proprie- hemoglobin l0 g/dl, and a normal PT/FIT prior to starting tary name for the L-threo enantiomer of dihydrosphingosine. therapy. Normal renal function (serum creatinine l.5 mg/dl) and The chemical name is (2S,3S)-2-amino-l,3-octadecanediol; the normal hepatic function (serum bilirubin, l.5 mg/dl; serum as- Eli Lilly Research Laboratory code number is SPC-100270; and partate aminotransferase and alkaline phosphatase levels 2.5 safingol is a white to off-white crystalline solid with a molecular times the upper limit of normal) were required. Patients with weight of 301.50. The molecular formula is C18H39N02. documented bone metastases and an elevated alkaline phosphatase Safingol was supplied by Eli Lilly Research Lab (Indian- >25 times the upper limit of normal were eligible for the study if apolis, Indiana) as a 0.5% (5 mg/mI) emulsion, which was all other liver function tests were within the above-specified limits. diluted with 5% dextrose in water to achieve a 0.5 mg/ml A normal resting gated pool heart scan with an ejection fraction solution for administration. The lipid emulsion contained soy- 50% was required. Patients were required to have no prior history bean oil, Pluronic F-68 (NF), cholesterol (NF), a-tocopherol of cardiac arrhythmia requiring therapy (other than chronic atrial (USP), glycerin (USP), water, and HC1. The vials were stored fibrillation). Patients with a history of a myocardial infarction under at 2-8#{176}Cand were for single-use only. The appropriate within 6 months prior to study entry were excluded. Prior DOX amount of safingol was diluted in 5% dextrose in water to treatment was allowed as long as the prior lifetime cumulative dose achieve the desired concentration. The diluted emulsion was ofDOX did not exceed 280 mg/m2. Good peripheral venous access stored at room temperature and was administered within 12 h of was required; otherwise central access via a Mediport or Broviac dilution when stored diluted in either polyvinyl chloride bags or catheter was required. Patients with evidence of hemolysis or a glass bottles. To ensure adequate mixing, the diluted emulsion history of non-drug-induced hemolysis (e.g., spherocytosis) were were shaken just prior to the start of administration. Adriamycin excluded. Females of child-bearing potential were required to have PFS, the brand of DOX manufactured by Pharmacia Laborato- had a negative serum pregnancy test and to use an acceptable ries (Columbus, Ohio), was supplied for use in this study. method of birth control (I.U.D., oral contraceptive, or barrier de- As shown in Fig. 2, the first cycle oftreatment for each patient vice). Patients without measurable disease were allowed entry on began with the administration of safingol alone over 1 h at the first this Phase I protocol although attempts were made to define meas- dosing visit (day I, dose 1). Two weeks later (day 14), ifthe patient urable disease in all patients so that the therapeutic efficacy of the did not have any ongoing toxicity, the patient received the second combination could be evaluated. This study was approved by the administration of safingol as a 1-h infusion at the same dose (dose Institutional Review Board at Memorial Sloan-Kettering. All pa- 2) over 1 h. Beginning 1 h after completion ofthe sa.fingol infusion, tients must have signed informed consent indicating that they were DOX was delivered via a 5-mm iv. push. aware of the investigational nature of the treatment. Because safingol emulsion had not previously been given The pretreatment evaluation included a complete medical to humans, the first six patients who received the first dose level history and physical examination including documentation and (15 mg/m2) were hospitalized for 24 h following their first two

Table I Hemolysis toxicity scale

Grade 0: No evidence of hemolysis

Grade 1: Laboratory evidence of hemolysis with any one of the following findings:

- reticulocyte index >2

- decrease (from baseline) in serum haptoglobin of 50% or more

- visual evidence of hemolysis in spun plasma (pink to purple change in color)

- positive finding of urine hemosiderin

- heme-positive urine without RBCs in the urinary sediment

- a blood smear (red cell morphology) suggestive of hemolysis

?it! no significant decrease in whole blood hemoglobin ( 10% decrease from baseline) titi no evidence of renal insufficiency (as defined below) Grade 2: Laboratory evidence of hemolysis (as above) and a 10. 1-20.0% decrease from baseline in whole blood hemoglobin or transient mild renal effects (creatinine of 1 .5-3.0 X the upper limit of normal) Grade 3: Laboratory evidence of hemolysis (as above) a 20. 1-30.0% decrease from baseline in whole blood hemoglobin or transient moderate renal insufficiency (creatinine of 3. 1-6.0 X the upper limit of normal) Grade 4: Laboratory evidence of hemolysis (as above) and a >30% decrease from baseline in whole blood hemoglobin or severe renal insufficiency (creatinine >6.0 X the upper limit of normal or requiring dialysis)

safingol treatments. In addition to pre-dose assessments at the administration of any cycle of treatment that resulted in a cumula- start of each cycle of treatment, vital signs were measured every tive life-time dosage of DOX of >300 mg/m2 and at the final study 15 mm during the safingol infusion, 1 h after completion of the evaluation visit. Gated pool heart scan was required prior to any safingol infusion (just prior to the administration of DOX), and cycle of treatment that resulted in a cumulative DOX dose of >300 hourly thereafter for the remaining observation period in the mg/m2. Tumor measurements with appropriate imaging studies clinic (up to 6 h post-safingol infusion, for the first two safingol were performed after each cycle of combination therapy. visits; up to 3 h post-safingol infusion at other cycle dosing Individual patients could receive the same dose of safingol visits). Physical exam, CBC with differential, platelets, PT, and DOX every 21 days unless there were signs of tumor progres- P’FT, biochemical screening profile, blood urea nitrogen, creat- sion, development of a significant toxicity that, in the investigator’s mine, electrolytes, and urinalysis were also obtained 24 h after judgment, precluded further therapy, clinical deterioration, or evi- safingol administration for the first two safingol dosing visits. dence of a decrease in left ventricular ejection fraction of 10% Because safingol administration was associated with he- from baseline. if toxicity resulting from the administration of safin- molysis in the animal models, extensive hemolysis assays were go! alone at the first dosing visit persisted for 2 weeks, patients required for all patients. These included: serum hemoglobin, were not eligible to receive the first administration of safingol in hematocrit, red cell morphology, serum haptoglobin, and urine combination with DOX. Depending on the nature of the toxicity, for hemosiderin prior to dosing at each dosing visit and at fixed the investigator could withdraw the patient from the study at that intervals during and following safingol administration. To pre- time or wait up to 2 weeks for the toxicity to resolve before vent mechanical hemolysis during blood drawing for these administering combination treatment. assessments, a 21-gauge (or larger) needle was used. In view of The dose of safingol received by a given patient was the lack of an National Cancer Institute common toxicity scale neither increased nor decreased during subsequent cycles. The for hemolysis, it was necessary to devise our own grading scale dose of DOX remain fixed (either at 45 or 60 mg/m2) for each (Table 1 ). If a patient developed hemolysis after a given cycle of patient regardless of safingol dosage. If the safingol infusion treatment in the study (including the administration of safingol was administered into a peripheral vein, DOX was injected into alone at the first dosing visit), a series of modifications to the a different peripheral vein. Both drugs could be administered subsequent administration(s) of safingol in that patient were into the same central line. The dose of safingol was sequentially implemented. Patients who developed grade I or greater hemol- increased in each cohort of three patients by 100%. If any one ysis through a central or peripheral vein would receive, on patient developed grade 2 toxicity of any type attributed to subsequent cycles, the safingol infusion only into a central vein safingol alone during the first 2 weeks, or if there was an at one-half the concentration and twice the volume of the prior apparent increase in the expected toxicity of DOX following regimen. All other nonhemolytic toxicities were graded accord- administration of the combination of safingol and DOX, or if the ing to the National Cancer Institute common toxicity criteria. serum levels of safingol approached those associated with tox- Additional visits during the first cycle of treatment were icity in preclinical animal studies, subsequent increases in the scheduled 1 week after dosing with safingol alone and at weekly dose of safingol were made according to a modified Fibonacci intervals for 3 weeks after dosing with combination treatment. scale. Each group of three patients was treated and followed for CBC and platelet count were obtained weekly during the course of 21 days after administration of combination treatment before the therapy. A chest X-ray was obtained each month. Compliance with next group of three patients was enrolled. ongoing birth control in female patients of child-bearing potential DLT was defined as the occurrence of grade 4 hematolog- was verified weekly and a serum pregnancy test, if indicated, was ical toxicity, grade 4 nausea and vomiting, grade 2 neurological obtained. A 12-lead EKG was performed 2-3 h after safmgol toxicity, or grade 3 toxicity of any other kind, including hemol- administration for the first three safingol treatments and before ysis. If DLT was demonstrated in one or two of three patients,

Table 2 Pat ients characteristics Patients received a mean of 2.5 cycles (range, 1-1 1) of the

No. of patients entered 17 safingol and DOX combination. At the 120 mg/rn2 level, one Median age (range) 59 (29-77) patient received 1 1 cycles of safingol (total safingol dose, 1320 Median KPS (range) 80 (70-90) mg/rn2) in combination with DOX (total DOX dose, 495 mg/rn2) Male : female 10 : 7 without evidence of cumulative hematological or cardiac toxic- Tumor types Pancreatic: 6 it)’. No patient required a dose reduction of either safingol or Gastric: 2 Colon: 3 DOX because of cumulative hematological toxicity at any dose Unknown primary: 2 level. Sarcoma: 3 An additional three patients were treated with 15 mg/rn2 of Nasopharyngeal: 1 safingol but with 60 mg/m2 of DOX (Table 3). There was no toxicity with safingol alone. However, all three patients experienced dose-limiting leukopenia (mean WBC nadir, 0.4 X l03/pA; range, 0.4-0.6 x 103/pJ) or neutropenia (mean absolute an additional three patients were enrolled at the same dosage. If neutrophil count nadir, 0.4 X 103/pJ; range, 0.1-0.9 X 103/ji.l) no more than two of all six patients encounter DLT, three with their first cycle of safingol and DOX together. There was patients were to be enrolled at the next higher dosage level. If also significant thrombocytopenia (mean platelet nadir, 55 X three or more of all six patients develop DLT, then the maxi- 10/.l; range, 30-90 x 103/pJ). Two of these three patients had mum tolerated dose was exceeded, and three more patients were been extensively pretreated with chemotherapy. treated at the next lower dose. The maximum tolerated dose was Nonhematological Toxicity. Nonhematological toxicity defined as the dose level at which 0 of 6, 1 of 6, or 2 of 6 patients was also mild and not dose-limiting with either safingol alone or experienced DLT, with the next higher dosage having at least 3 in combination with DOX. One patient at a safingol dose of 60 of 6 patients encountering DLI. mg/rn2 experienced (when safingol was administered alone) Pharmacokinefics. Pharmacokinetics were performed at grade I hernolysis as evidenced by a >50% decrease in serum each of the first two safingol dosing visits. Plasma samples were haptoglobin. This patient’s haptoglobin levels decreased from a obtained for HPLC analysis at the following time points in baseline of 1 10 mg/dl to 26 rng/dl 6 h following safingol therapy minutes: 60 (just prior to the start of the safingol infusion), 0 and was fully recovered 72 h later. There was no associated (immediately after completion of the 1 h of safingol infusion), 5, change in reticulocyte count, no evidence of hemolysis on the 15, 30, 60, 65, 80, and 95 minutes and then 1, 2, 3, 4, 6, and 24 h peripheral smear, no pink discoloration to the plasma, and no after safingol infusion. evidence of urine hemosiderin. This degree of hernolysis was The method for safingol measurements requires HPLC with believed to be related to this patient’s relatively poor venous fluorescence detection. Briefly, safingol was extracted with N-butyl access. The patient was retreated on day 14, according to the chloride from human plasma made basic by the addition of 0.1 N protocol criteria for grade I hernolysis, which included infusion sodium hydroxide. The organic layer was transferred to a clean test of safingol through a central vein at one-half the concentration tube and evaporated to dryness. The residue was reconstituted in (e.g., twice the total volume but the same total dose). Under methanol and derivatized with o-phthaldialdehyde in the presence these conditions, the patient experienced no further evidence of of mercaptoethanol for HPLC analysis (12). This method is sensi- hemolysis. One patient registered to the study with a diagnosis tive to a safingol concentration of 0.036 p.g/ml. The method for of adenocarcinorna of unknown primary developed intense fa- DOX measurements was performed as described previously (13). cial flushing, without other associated toxicity, after receiving The ensuing plasma concentration-time profiles were generated only 2-3 mg of safingol. She was taken off study and rendered using a noncompartmental method (14). inevaluable for response. Initially, this was believed to be an allergic reaction to safingol that was unrelated to the total

RESULTS safingol dose. Later, the patient had an ‘‘ ‘In-pentatreotide scan

Patient Population As shown in Table 2, 17 patients that was highly positive for somatostatin receptor-positive 1ii entered the study (10 males and 7 females). The median age of mor at the sites of her multiple liver metastases. She, therefore, this group was 59 (range, 29-77), and the median Karnofsky was subsequently adjudicated as having a neuroendocrine tu- performance scale was 80 (range, 70-90). There was a wide mor, and the facial flushing was related to her underlying range of tumor types entered into the study. These include: disease rather than directly related to the drug. pancreatic (6), gastric (2), colon (3), unknown primary (2), Safingol Pharmacokinetics. Fig. 3 shows a plot of Cmax sarcoma (3), and nasopharyngeal (1). Twelve patients had been and AUC for each patient versus safingol dose in mg/rn2. As treated previously with chemotherapy. shown, up to a safingol dose of 120 mg/rn2, the increase in Cmax Hematological Toxicity. Safingol either alone (data not and AUC was generally linear with increasing safingol dose. At shown) or in combination with 45 mg/m2 DOX (Table 3) did not 120 mg/rn2, the mean Cmax was 1040 ± 196 ng/ml, and the cause dose-limiting hematological toxicity. The dose escalation mean AUC was 1251 ± 317 ng X h/rnl. There were excellent of safingol with 45 mg/m2 of DOX was carried out to a safingol correlations of 0.82 and 0.87 between the safingol dose in dose of 120 mg/rn2 before the trial was suspended for lack of mg/m2 and the respective Cmax and AUC of the drug. sufficient drug supply. As shown on Table 3, with the combi- Analysis of the AUCs for DOX indicate that increasing nation of 120 mg/rn2 of safingol and 45 mg/rn2 of DOX, the doses of safingol did not affect the AUC of a 45 mg/rn2 iv. mean WBC, absolute neutrophil count, and platelet counts were bolus of DOX (Table 4). For example, the mean AUC of DOX 5.4 X 1034a1, 5.5 X l03/i.l, and 263 X l034t1, respectively. (1320 ± 260 ng X h/mI) with 30 mg/rn2 of safingol was not

Table 3 Hemato logical toxicity with sa fingol and DOX

Safingol DOX No. of Total no. of Mean nadir WBC X l0 Mean nadir ANC X iO Mean platelet X l0 (mg/m2) (mg/m2) patients cycles administered (range) (range) (range) 15 60 3 7 0.4 (0.4-0.6) 0.4 (0.1-0.9) 55 (30-99) 15 45 3 3 3.29(1.4-5.0) 1.2(0.6-1.6) 150(121-196) 30 45 4 7 5.9 (3.1-9.0) 3.0 (2.0-4.0) 236 (150-349) 60 45 3 8 4.0 (2.2-6.1) 2.3 (0.7-4.7) 154 (70-233) 120 45 3 15 5.4 (4.9-7.5) 4.5 (2.3-5.1) 263 (225-279)

A Table 4 Effect of safingol on DOX (45 mg/rn2) AUC 1400 . Safingol dose No. of Mean DOX AUC 1200 (mg/m2) patients (ng X h/ml, ± SD) I - 7.75(x) + 51.81 30 3 1320(± 260) 1000 082 60 3 1451 (± 635) E 120 3 ll00(± 310)

600 I C.) 400 Table 5 Eff ect of DO X (45 mg/m2) on safin gol pharmacokinetics

200 Mean safingol Mean safingol Safingol dose No. of AUC without DOX AUC with DOX (mg/rn2) Patients (ng X mg/ml, ± SD) (ng x mg/mI, ± SD) ) 25 50 75 100 125 15 3 185±94 151±84 Safingol Dose, mg/rn2 30 3 232±78 353±260 60 3 557±61 450±24 B 120 3 1251 ± 317 1226 ± 262 1600 . 1400 . 1200 I Y 10.25(xl + 14.#{243}j E k’0.87 I ‘: 1000 765 rnl/min, and the mean volume of distribution was 995 ± . 421 liters (Fig. 4A). These values were not appreciably different C than those obtained for 120 mg/rn2 of safingol (dose 2) co- 600 S I administered with 45 mg/rn2 of DOX: mean half-life, 4.39 ± I 400 3.4 h; mean clearance, 3147 ± 761 mI/mn; and mean volurne of distribution, 1183 ± 935 liters (Fig. 4B). With safingol alone, 200 two patients (nos. 15 and 17) exhibited an increase in their 0 safingol plasma concentrations 3-4 h after completion of the 0 25 50 75 100 125 infusion (Fig. 4A). This apparent inflection may represent en- Safingol Dose, mg/rn2 tero-hepatic recirculation, or, because safingol is lipophilic, it

Fig. 3 Pharmacokinetic dose-ranging for Cmax (A) and AUC (B) of could indicate release of safingol frorn fat stores. However, safingol. Cmax (ng/ml) and AUC (ng X h/ml) are shown for each patient because these changes were not evident when safingol was studied and plotted against safingol dose (mg/m2). r estimate of administered with DOX (Fig. 4B), the basis for the change in the variability. The equations shown represent the best fit regression line shape of the C x t is unclear and awaits further investigation. from the data. The results indicate that the increase in Cmax and AUC is Clinical Response to Safingol and DOX. We have ob- linear with increasing safingol dose. served signs of clinical activity in several patients. One patient with pancreatic cancer treated at a safingol level of 15 mg/rn2 had a 30% decrease in the size of a measurable omental metas- significantly different from the mean AUC of DOX (1 100 ± 310 tasis (from 41 cm2 to 26 cm2). In addition, she had a decrease in ng X h/mi) with 120 mg/rn2 ofsafingol. In addition, the mean AUC biochemical markers associated with her disease. These in- for safmgol did not change with the coadministration of a fixed cluded a decrease in CA 19-9 from 3300 units/rnl to 2200 dose of DOX. For example, as shown in Table 5, at the 120 mg/rn2 units/mi and a decrease in CA-l25 from 428 units/rnl to 220 safingol level, the mean AUC for safingol without DOX was units/ml. A second patient with pancreatic cancer at a safingol 1251 ± 317 ng X mg/mi, whereas the mean AUC for safingol with dose of 15 mg/rn2 experienced a decrease in the size of his 45 mg/rn2 of DOX was 1226 ± 262 ng X mg/mi. primary pancreatic neoplasm and decreases in biochemical The data from the three patients treated at the 120 mg/rn2 markers with a CA 19-9 decreasing from 12,000 units/mi to level were analyzed according to a noncompartmental method. 9,000 units/ml and a carcinoernbryonic antigen decreasing from For dose 1 (safingol without DOX), the mean safingol half-life 130 ng/ml to 100 ng/ml. A third patient with pancreatic cancer was 3.97 ± 2.51 h, the mean estimated clearance was 3140 ± at the 60-mg/rn2 safingol level experienced clinical improve-

A had been shown in animals not to cause such toxicity, and, there- Safingol, 120 mg/rn2: Dose I fore, appeared to be an attractive agent for clinical development 2000 (20, 21). 1000 The starting dose of safingol was selected as 15 mg/rn2.

E This dose represented one-tenth of the mouse LD10, 4% of the I Pt. 15 0 HNTD in dogs, and 12% of the HNTD in rats. These HNTDs for C Pt. 16 : 100 ______I Pt. 17 dogs and rats did not increase the toxicity of minimally or U C 0 moderately toxic doses of DOX in these species (1 1). The study C.) design, though, did allow us to assess the acute toxicity of safingol as a 1-h infusion administered 2 weeks before the a! combination with DOX. This clinical trial has shown that safingol, up to a dose of 120 mg/rn2, is well tolerated both by itself and when administered in combination with 45 mg/rn2 of DOX.

-4 4 8 12 16 20 24 There was no dose-limiting hematological toxicity. Time, hours From the mice studies with safingol, it was reported that to achieve meaningful chemopotentiation with DOX, it was nec- B essary to deliver a safingol dose of at least 5 mg/kg (9). From the 3000 Safingol, 120 mg/rn Doss 2 animal studies, a single 5 mg/kg i.v. dose of safingol resulted in 2000 a mean plasma Cmax and AUC of 1797 ng/mJ and 668 ng X 1000 h/rnl, respectively (20). In our clinical trial at the highest dose E ______I Pt. 15 of safingol evaluated (120 mg/rn2), the mean Cmax was 988 C 0 Pt. 16 ng/rnl, and the mean AUC was 1277 ng X h/mI. Thus, when j 100 ______U Pt. 17 C 0 compared to the pharmacological data obtained at the 5-mg/kg C.) dose in the rodents, in this Phase I study we were able to E approach a therapeutic Cm,,,, for safingol as a chemopotentiating : a- agent for DOX and, in fact, had exceeded the lower theoretical, therapeutic AUC for this drug. More definitive resolution of this pharmacological end point awaits renewal of drug supply.

-4 0 4 8 12 16 20 24 The hernatological toxicity encountered in the three pa- Tirne, hours tients treated with 15 mg/rn2 safingol and 60 mg/rn2 of DOX was unexpected and was probably related directly to the dose of Fig. 4 Pharmacokinetics of safingol for the three patients treated at 120 DOX rather than to an increase in the toxicity of DOX by mg/m2 as a single agent (dose 1) or in combination with DOX (dose 2). safingol. In preclinical testing, safingol had no effect when tested as a single agent on colony-forming ability of normal human bone marrow progenitor cells in vitro at concentrations ment with radiological evidence of a decrease in ascites and stabi- 2-3-fold greater than that required to inhibit tumor cell prolif- lization of a previously rising carcinoernbryonic antigen. A fourth eration by 50%. In addition, when tested in combination, safin- patient at the 120-mg/rn2 level with angiosarcorna of the scalp and gol did not potentiate the cytotoxicity of doxorubicin or cisplatin lung metastases experienced flattening and shrinkage (<50%) in on normal bone marrow progenitor cells (11). Although this the size of his scalp lesions. This was associated with decreased study was not designed to study the pharmacokinetics of DOX head bleeding and a subjective improvement in cough. alone without safingol, it appears that the pharmacokinetics of 45 mg/rn2 of DOX was not affected by safingol, despite increas- DISCUSSION ing safingol concentrations. In addition, the pharmacokinetics Safingol, the L-threo enantiomer of dihydrosphingosine, is the for the 60-mg/rn2 dose of DOX at this level revealed a mean first PKC-specific inhibitor to enter clinical trials. Members of the AUC of 1680 ng X h/mi that did not substantially exceed the PKC family are characterized by a unique NI-12-terminal regulatory AUC associated with identical DOX doses in a published series domain containing cofactor binding sites and a COOH-terminal (13, 22). Therefore, we believe that the rnyelosuppression ob- catalytic domain that is homologous to that of these other protein served with 15 and 60 mg/rn2 of DOX was due to poor bone kinases (15, 16). Sphingosines have been shown to inhibit PKC marrow reserve in a predominantly pretreated patient population activity and phorbol myristate acetate binding by interfering with with advanced rnetastatic disease. This hypothesis will eventu- the function of the unique regulatory domain of PKC (17). This is ally require further testing. in contrast to the “classic” PKC inhibitor, staurosporine, which Although venous irritation and hemolysis were significant inhibits PKC activity by interacting with the catalytic domain of the and, in some instances, there were dose-limiting toxicities in the enzyme (18). However, because the catalytic domain of PKC is animal studies, up to a safingol dose of 120 mg/rn2, both as a highly homologous to the catalytic domain of other protein kinases single agent and in combination with 45 mg/rn2 of DOX, no that are critical for normal cellular function (i.e., pp60 tyrosine kinase and cyclic AMP-dependent protein kinase A) staurosporine has proven exceptionally toxic (19). Safingol, on the other hand, in

view of its specificity for the unique regulatory domain of PKC, 3 Unpublished studies from Eli Lilly Research Labs.

dose-limiting hemolysis was seen. Data from the predinical studies 5. Persons, D. A., Wilkison, W. 0., Bell, R. M., and Finn, 0. J. Altered indicated a hemolytic potential to safingol. This was determined growth regulation and enhanced tumorigenicity of NIH 3T3 fibroblasts not to be related to total safingol dose. It was demonstrated that transfected with protein kinase C-I cDNA. Cell, 52: 447-458, 1988. these effects were mitigated by the use of low safingol concentra- 6. Schwartz, G. K., Haimovitz-Friedman, A., Dhupar, S. K., Ehleiter, D., Maslak, P., Loganzo, F., Kelsen, D. P., Fuks, Z., and Albino, A. P. tions and large-sized, high flow veins. By following guidelines that Potentiation of apoptosis by treatment with the protein kinase C specific incorporated these observations, we witnessed only one grade I inhibitor safingol in mitomycin-C treated gastric cancer cells. J. NatI. toxicity. This was manifested as a transient fall in serum haptoglo- Cancer Inst., 8: 1394-1399, 1995. bin following infusion through a poor peripheral vein. This, in fact, 7. Sachs, C. W., Safa, A., Harrison, S., and Fine, R. L. Partial inhibition was prevented with subsequent administration of safingol in the of multidrug resistance by safingol is independent of modulation of same patient through a central venous catheter. Therefore, the P-glycoprotein substrate activities and correlated with inhibition of protein kinase C. J. Biol. Chem., 270: 26639-26648, 1995. hemolysis induced by safingol in the animal models can be avoided when administered along strict guidelines and, if needed, through a 8. Adams, L. M., Cofield, D. J., Seldin, J. C., Kitchen, P. A., Harrison, S. D., and Darges, J. W. Effect of the protein kinase C (PKC) inhibitor central vein in a more dilute concentration. SPC-l00270 on drug accumulation and cytotoxicity in drug resistant and We observed no major organ toxicity. Renal toxicity, con- sensitive tumor cells in vitro. Proc. Am. Assoc. Cancer Res., 34: 410, 1993. sidered a consequence of intravascular hemolysis, was observed 9. Adams, L. M., Dykes, D., Harrison, S. D., Saleh, J., and Sash, L. in rats and dogs (21) but was not observed in the clinical trial. Combined effect of the chemopotentiator SPC- 100270, a protein kinase At the 120-mg/rn2 level, one patient received a cumulative DOX C inhibitor, and doxorubicin or cisplatin (Cis) on murine isografts and dose of 400 mg/rn2 without change in his left ventricular func- human tumor xenografts. Proc. Am. Assoc. Cancer Res., 34: 410, 1993. tion, indicating no enhancernent of DOX-induced cardiac tox- 10. Siemann, D. W., Jiang, J. B., Ballas, L., and Janzen, W. Threo- dihydrosphingosine potentiates the in vito antitumor efficacy of cis- icity by safingol. Another concern was hepatic toxicity. Dogs platin and Adriamycin. Proc. Am. Assoc. Cancer Res., 34: 411, 1993. administered 40 mg/kg of safingol developed clinical pathology 1 1. Susick, R. L., Bozigian, H. P., Kurtzberg, J., Adams, L. M., Weiler, indicative of hepatobiliary involvement (2 1 ). Pharmacokinetic M. S., Harrison, S. D., and Kedderis, L. B. Combination toxicology studies assessment of these animals at this dose level indicated a mean with the chemopotentiating agent SPC-100270 (a PKC inhibitor) and che- Cmax of 9,033 ng/rnl and a mean AUC of 1 1 ,094 ng X h/mI. At motherapeutic agent. Proc. Am. Assoc. Cancer Res., 34: 410, 1993. the highest dose level of safingol ( 1 20 mg/rn2) evaluated in this 12. Rajewsky, R. A., Kosednar, D. G., Matches, T. A., Wong, 0. S., clinical study, the mean Cmax (1041 ng/ml) and mean AUC Burchett, K., and Thakker, K. Stereo-specific analysis of a novel protein (125 1 ng X h/rnl) were considerably below these levels and may kinase C inhibitor. J. Pharm. Biomed. Anal., 13: 247-253, 1995. account for the absence of observed hepatic toxicity. 13. Pierce, R. N., and Jatlow, P. I. Measurements of Adriamycin (doxorubicin) and its metabolites in human plasma using reversed-phase high This study shows that doses of safingol up to 120 mg/rn2 performance liquid chromatography and fluorescence detection. J. Chro- can be administered alone or in combination with 45 mg/rn2 of matogr., 164: 471-478, 1979. DOX without significant toxicity. Although signs of clinical 14. Gibaldi, M., and Pemer, D. Pharmacokinetics, Ed. 2, pp. 409-416. activity were observed, in terms of clinical design, it is not New York: Marcel Dekker, 1982. possible to definitively state whether this may have been from 15. Kikkawa, U., Kishimoto, A., and Nishizuka, Y. The protein kinase DOX alone and not from the combination therapy. The levels of C family: heterogeneity and its implications. Annu. Rev. Biochem., 58: safingol achieved in the plasma of patients at the highest dose 31-44, 1989. level of safingol tested approach the micrornolar concentrations 16. Coussens, L., Parker, P. J., Rhee, L., Yang-Feng, T. L., Chen, E., Waterfield, M. D., Franke, U., and Ullnch, A. Multiple, distinct forms of safingol that inhibit PKC in vitro and in vivo. Thus, this study of bovine and human PKC suggests diversity in cellular signaling suggests that the PKC inhibitor safingol, which inhibits signal pathways. Science (Washington DC), 233: 859-866, 1986. transduction pathways and potentiates DOX in vivo, can be 17. Hannun, Y. A., Loomis, C. R., Merrill, A. H., Jr., and Bell, R. M. safely administered to patients at systemic levels associated with Sphingosine inhibition of protein kinase C activity and of phorbol PKC inhibition as well as with chemopotentiation. dibutyrate binding in vitro and in human platelets. J. Biol. Chem., 261: 12602-12609, 1986. ACKNOWLEDGMENTS 18. Nakano, H., Kobayashi, E., Takahashi, I., Tamaoki, T., Kuzuu, Y., and Iba, H. Staurosporine inhibits tyrosine-specified protein kinase We acknowledge the participation of the Chemotherapy Research activity of Rous sarcoma virus transforming protein. J. Antibiot. (To- Nurses of Memorial Hospital for their attention to the detail in admin- kyo), 40: 706-708, 1987. istenng safingol and DOX, and the data management team. 19. Tamaoki, T., and Nakano, H. Potent and specific inhibitors of PKC or microbial origin. Biotechnology, 8: 732-735, 1990. REFERENCES 20. Bozigian, H., Teo, S., Kleeman, J., Brisson, J., and Harrison, S. D. I. Kikkawa, U., and Nishizuka, Y. The role of protein kinase C in Comparison of the pharmacokinetics of SPC-l00270, a protein kinase C transmembrane signalling. Annu. Rev. Cell Biol., 2: 149-178, 1986. inhibitor in mice, following administration of toxic and therapeutic 2. Bell, R. M. Protein kinase C activation by diacylglycerol second doses. Proc. Am. Assoc. Cancer Res., 34: 410, 1993. messengers. Cell, 45: 631-632, 1986. 21. Kedderis, L. B., Weiler, M. S., Christian, B. J., Thomford, P. J., 3. Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Hall, R. L., Salamon, C. M., Harrison, S. D., and Susick, R. L. Preclin- Bell, R. M. Quantitative measurement of sn-l ,2-diacylglycerols present ical safety assessment of SPC- 100270 emulsion, a novel protein kinase in platelets. hepatocytes and ras- and sis-transformed normal rat kidney C inhibitor under development for the use as a chemopotentiation agent. cells. J. Biol. Chem., 261: 8597-8600, 1986. Proc. Am. Assoc. Cancer Res., 34: 410, 1993. 4. Smart, R. C., Huang, M. T., and Conney, A. H. sn-l,2-Diacylglyc- 22. Jacquet, J-M., Bressolle, F., Gaitier, M., Bourrier, M., Donadio, D., erols mimic the effects of l2-O-tetradecanoylphorbol-13-acetate in vivo Jourdan, J., and Rossi, i-F. 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Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 1997 American Association for Cancer Research. A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin.

G K Schwartz, D Ward, L Saltz, et al.

Clin Cancer Res 1997;3:537-543.

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