A Phase I Study of Swainsonine in Patients with Advanced Malignancies1

[CANCER RESEARCH 54, 1450-1457, March 15, 1<Â»4] A Phase I Study of Swainsonine in Patients with Advanced Malignancies1

Paul E. Goss2, Jose Baptiste, Berny Fernandes, Michael Baker, and James W. Dennis3

Toronto Hospital, Department of Medical Oncology, Faculty of Medicine Â¡P.E. G., M. B.Â¡,and Department of Molecular & Medical Genetics [J. B., J. W. Â£>./,University of Toronto; and Samuel Lunenfeld Research Institute, Mount Sinai Hospital [B. E, J. W. Â£>./,Toronto, Ontario, M5G 1X5

ABSTRACT Furthermore, inhibitors of JV-linked oligosaccharide processing such as castanospermine and swainsonine have been shown to attenuate Swainsonine, an a-mannosidase inhibitor which blocks Golgi oligosac- both metastasis and tumor growth in animal models (8-11). charide processing, represents a new class of compounds that inhibit both Swainsonine is an indolizidine alkaloid found in Australian Swain- rate of tumor growth, and metastasis, in murine experimental tumor models. In this first phase I study, the quantitative and qualitative toxici- sona canescens (12), North American plants of the genera Astragalus ties of Swainsonine have been studied in patients given a continuous i.v. and Oxytropis (13), and also in the fungus Rhizoctonia leguminocola infusion over 5 days, repeated at 28-day intervals. Dose levels were esca (14). The alkaloid is a potent inhibitor of the Golgi enzyme a-man lated in increments of 100 Â¿ig/kg/day from 50-550 jug/kg/day. Nineteen nosidase II (15), an enzyme required for maturation of TV-linked oli patients with both solid tumor and hematological malignancies were given gosaccharides on newly synthesized glycoproteins. Swainsonine also a total of 31 courses. Hepatotoxicity, particularly in patients with liver blocks lysosomal a-mannosidases causing the accumulation of oligo mÃ©tastases,was the dose-limiting toxicity. The maximum tolerated dose mannoside chains in cells exposed to the drug (16). T24H-ras-trans- iMTDi and the recommended starting dose (MTD -1 level) were 550 and fected NIH 3T3 cells lose their ability to grow in an anchorage- 450 /ig/kg/day, respectively. Common side effects included edema, mild independent manner when cultured in the presence of swainsonine liver dysfunction, a rise in serum amylase, and decreased serum retino!. (17), suggesting induction of a more differentiated phenotype. The Acute respiratory distress syndrome possibly precipitated by swainsonine autocrine-dependent growth rate of murine and human tumor cell lines resulted in a treatment-related death in a patient with significant pretreat in vitro is reduced by swainsonine (18, 19). Swainsonine-treated ment hepatic dysfunction. One patient with head and neck cancer showed >50% shrinkage of tumor mass for 6 weeks after treatment. Two patients MDAY-D2 lymphoma tumor cells show reduced adhesion to endo- with lymphangitis carcinomatosis on chest X-ray noted improvement in thelial cells in vitro and in vivo (20), and swainsonine blocks invasion cough and shortness of breath during the infusion of swainsonine and for of murine and human tumor cells through extracellular matrix while 1 week thereafter. enhancing cell adhesion to extracellular matrix proteins in vitro Clearance and serum half-life for swainsonine were determined to be (21, 22). approximately 2 ml/h/kg, and 0.5 day, respectively. Golgi oligosaccharide In animals, swainsonine exhibits antitumor activity when given p.o. processing, a putative anticancer target for swainsonine was inhibited in (8, 23, 24), by i.p. injection (25), and by systemic infusion (18). For peripheral blood lymphocytes as evidenced by a marked decrease in leu- example, swainsonine administered to athymic nude mice in drinking koagglutinin binding after 5 days of treatment. Oligomannosides in pa tient urine increased 5-to 10-fold over the 5 days of treatment, indicating water inhibited the growth rate of human colorectal carcinoma and that tissue lysosomal a-mannosidases were also blocked by swainsonine. MeWo melanoma tumor xenografts by 50% (18, 19). The growth of Urine oligomannoside accumulation reached steady state at 3 days, ap murine Sarcoma 180 ascites tumors was completely inhibited by 30 proximately 1 day after serum drug levels reached steady state. The and 100 mg/kg/day administered i.p. for 5 consecutive days after fraction of HLA-DR-positive cells in peripheral blood lymphocytes in tumor cell inoculation (25). Swainsonine p.o. at 10 /J-g/ml in the creased following 5 days of swainsonine treatment, an effect similar to that drinking water of athymic nude mice showed additive antitumor ac observed for peripheral blood lymphocytes from normal subjects cultured tivity with a interferon, such that human HT29m colon carcinoma with swainsonine. No significant changes in CD3, CD4, CDS, CD16, and xenografts grew at one-tenth the rate of those in untreated mice (18). CD25 were observed. Swainsonine produces minimal toxicity when ad The action of swainsonine as an immune modulator has been re ministered i.v. to cancer patients at dosages that inhibit both Golgi a-man viewed (23, 26). When given p.o. to mice, the alkaloid alleviates nosidase II and lysosomal a-mannosidases. Detection of hepatic mÃ©tasta chemically and tumor-induced immune suppression (25, 27, 28), ses or liver enzyme abnormalities prior to treatment predict for more stimulates lymphocyte proliferation (29) and NK4 cell activity (23), significant toxicity. and activates macrophages (30, 31). The drug also stimulates LAK cell activity when added to cultures of human lymphoid cells (32). Preclinical toxicology studies in rodents showed swainsonine to be INTRODUCTION well tolerated (18, 33, 34). No mortality occurred in young rats given Malignant transformation in rodent and human tumors is associated swainsonine at doses up to 46 mg/kg/day by miniosmotic pump for with increased branching at the trimannosyl core of complex-type periods of 3 weeks (34). The drug suppressed the growth rate of young asparagine (A^-linked oligosaccharides (1-3). We have shown that the rats apparently by suppression of appetite, and caused lysosomal degree of ÃŸ1-6branching in rodent tumor models (3, 4) and in human storage of oligosaccharides in tissues including the central nervous breast and colon carcinomas (5, 6) correlates with disease progression. system. However, no evidence of neurotoxicity was noted in this or More importantly, we have demonstrated that somatic mutations other preclinical studies on rodents treated with swainsonine (19, 34). which inhibit oligosaccharide processing in the Golgi dramatically In this first phase I study, cancer patients were given courses of inhibit mÃ©tastasesand reduce solid tumor growth in mice (4, 7). continuous 5-day i.v. infusions of swainsonine in escalating doses from 50-550 /xg/kg/day repeated every 28 days. The starting dose level of 50 pig/kg/day was an estimate of the p.o. dosage which had Received 8/9/93; accepted 1/14/94. The costs of publication of this article were defrayed in part by the payment of page antitumor activity in mice (18, 19, 23). The schedule of a 5-day charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by research grants from National Cancer Institute of 4 The abbreviations used are: NK, natural killer; LAK, lymphocyte-activated killer: Canada to J. W. D., and by a grant from the Mount Sinai Hospital for the purchase of AST, aspartate aminotransferase; MTD, maximum tolerated dose; FACE, fluorphore- swainsonine. assisted carbohydrate electrophoresis; FACS, fluorescence-activated cell sorter; PEL, 2 To whom requests for reprints should be addressed, at The Toronto Hospital. MLW peripheral blood lymphocytes; L-PHA, phylohemagglutinin; IL, interleukin; TNF, tumor 2-022, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4. necrosis factor; FITC, fluorescein isothiocyanate; ALT, alanine aminotransferase; Glc- 3 Senior Research Scientist of the National Cancer Institute of Canada. NAc, /V-acetylglucosasmine; Man, mannose. 1450

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1994 American Association for Cancer Research. SWAINSONINE IN ADVANCED MALIGNANCIES infusion given every 28 days was based primarily on the antitumor of nitrogen at 60Â°C.The residue was further dried in a desiccator over P2O5 at activity noted in mice given a 24-h exposure to swainsonine in an room temperature for 12 h. interrupted twice weekly schedule (24). The limitations of adminis To the dry samples were added 1 ml of dry acetonitrile, 15 mg of 4-dimeth- tering an in-hospital 5-day infusion to patients with advanced malig ylaminopyridine, and 15 mg of sodium acetate. The samples were cooled to 0Â°Cand 0.5 ml of acetic anhydride was added and left overnight at room nancies more often than once monthly were also taken into consider temperature. The acetylated reaction products were then partitioned between 1 ation. The data on toxicity, serum drug levels, Golgi and lysosomal a-mannosidase inhibition, and lymphocyte marker changes are in ml of chloroform and 1 ml of distilled water with gentle shaking of the samples. Polar contaminants dissolve in the aqueous layer, whereas the acety cluded in this report. lated saccharides and swainsonine dissolve in the lower chloroform layer. The chloroform layer was washed with 1 ml of water, and traces of water and acetic anhydride were removed by passing the samples over 500-mg cartridges of basic alumina (Alltech Inc.). The solvent was evaporated at 60Â°Cunder a PATIENTS AND METHODS stream of nitrogen, the residue was dissolved in 30 JA!of chloroform and Patients. Nineteen patients were enrolled in the study between January injected in l-^il aliquots into the gas-liquid chromatogram. 1992 and June 1993. The study was approved by the University of Toronto and Gas-liquid Chromatographie analysis of each derivatized sample was done The Toronto Hospital ethics committee. Patients aged 18-75 years with a life by using a Model 3400 Varian Gas Chromatograph equipped with flame expectancy of at least 3 months, Eastern Cooperative Oncology Group perfor ionization detector and wall coated open tubular fused capillary column (15 m mance status 0-3, and histologically confirmed diagnosis of metastatic carci x 0.53 mm inside diameter; Megabore) coated with 1.2-nm thin layer of noma, leukemia, or lymphoma were eligible for enrollment on study. No DB225. The column temperature was kept at 180Â°Cfor 22 min after the recognized alternative therapeutic options were available for study patients. injection, then raised from 180 to 230Â°Cat lÂ°C/min; the latter temperature Exclusion criteria included significant cardiac (congestive heart failure or was maintained for 30 min. The injector port temperature was 240Â°C,and the refractory edema), hepatic (bilirubin >34 pimol/liter or AST > 2x normal), detector block was 280Â°C. Samples of 1 jil were injected by direct flash renal (creatinine >175 /xmol/liter), or neurological impairment. Screening prior vaporization, and prepurificd dry helium (Canox) was used as the carrier to enrollment on study included: signed informed consent; history and physical gas at a flow rate of 6 ml/min. Quantitative analyses were done by using a and assessment of Eastern Cooperative Oncology Group performance status; Model 3396B Hewlett-Packard integrator. Recovery of swainsonine was 90% chest X-ray, routine hematology, biochemistry, urinalysis and electrocardio; and the standard deviation for reproducibility on 10 replicate samples was arterial blood gas determination and resting respiratory function testing were 5%. added to the pretrcatment assessment after edema was noted in 2 of 3 patients Urine Oligomannosides. FACE from GLYKO was used for determining treated at level 1. Base-line symptoms were recorded. swainsonine-induced oligosaccharide storage. The reducing end of saccharides All patients were examined twice daily, including a neurological assess are subject to reductive amination with 7-aminonapthalene-l,3-disulfonic acid ment, and all screening blood work was repeated daily. Arterial blood gas (36). Five-fil samples of urine were dried, and to each tube were added 5 jj.1 determination, respiratory function testing, and chest X-ray were repeated once of 0.2 M7-aminonapthalene-l,3-disulfonic acid in 2.6 Macetic acid and 5 /il of between days 2 and 4 during the 5-day infusion. Patients were seen as out NaCNBH, in l Mdimethyl sulfoxide, followed by incubation at 37Â°Cfor 2 h. patients 3 weeks after each course of swainsonine for a full assessment and After drying, samples were redissolved in 62.5 IHMTris-HCl, pH 6.8, contain decision regarding their next course of treatment. ing 20% glycerol, and were applied to 30% polyacrylamide gels, precast by Swainsonine Administration. Swainsonine was synthesized by Toronto GLYKO. Gels were run at 15 mA constant current and cooled to 4Â°Cfor 2 h. Research Chemicals and purified by recrystallization to constant melting point, The separated oligosaccharides were imaged with a high-sensitivity digital and purity was confirmed by proton nuclear magnetic resonance spectroscopic camera and computer system. This method allows less than 1 pmol of oligo analysis to be greater than 98%. saccharide to be detected and quantified by comparing test samples to Each course of swainsonine was administered to patients by pump as a standards run in parallel. continuous infusion via a peripheral vein over 5 days and repeated at 28-day FACS Analysis of PBL. PBL were prepared and analyzed for CD3, CD4, intervals. The daily dose was prepared immediately prior to use in one liter of CDS, CD14, CD16, CD25, CD57, and HLA-DR by FACS. Peripheral blood 0.9% saline solution and the bag was protected from light during the infusion. was collected in heparin-containing tubes, transported at room temperature, Blood for laboratory testing was taken from a heparinized indwelling catheter and analyzed within 48 h. Monoclonal antibodies conjugated with either in the opposite arm. The dose levels were 50, 150, 250, 350, 450, and 550 FITC or phycoerythrin were purchased from Becton-Dickinson: FITC-labeled fig/kg/day. L-PHA was from E.Y. Labs. Aliquots of 100 /xl of whole blood were incu Dose Escalation. Three patients were treated at each dose level. Dose bated with anti-CD antibodies or 0.1 /xg/ml of FITC-labeled L-PHA for 20 escalation occurred until dose limiting toxicity (MTD) was reached. MTD was defined as <3 of 3 patients experiencing grade 3, and 0 of 3 patients grade 4 min followed by lysis of RBC, washing by centrifugation, and fixation of the nonhematological or hematological toxicity according to standard National cells by using the automated 0 Prep system (Coulter). The lymphocyte popu Cancer Institute of Canada toxicity criteria after one course of treatment. The lation was gated, and the contaminating monocytes were assessed by staining exception was grade 4 edema in patients with preexisting edema which was not with CD 14 which was consistently below 1% of the gated population. Cytokine Measurements. IL-6 and TNF-a levels in patient serum were considered a defining criterion for MTD. Intrapatient dose escalation was not allowed. In the event of MTD being reached, 3-further patients were to be measured by using enzyme-linked immunosorbent assay kits (R&D). The treated at 1 level lower until no more than 2 of 6 patients experienced grade 3 cytokine enzyme-linked immunosorbent assay test kit is a solid-phase enzyme or greater toxicity. This level would define the "recommended starting dose" immunoassay using capture and detection antibodies, and was used as de for future clinical testing. scribed by the manufacturer. Serum Drug Levels. The method for extraction, acetylation, and measure Serum Retino! Measurements. Serum rctinol levels were measured as ment of serum swainsonine is described in detail elsewhere (35). Briefly, the previously described (37). Briefly, 1-ml serum samples were spiked with internal standards methyl a-n-mannopyranoside and methyl ÃŸ-u-galactopyr- retinyl acetate and extracted with 0.4 ml of equal volumes of butanol/ anoside were prepared in H;O at 100 fig/ml, and 45 jj.1of each standard were acetonitrile, then 0.3 ml of 1.2 kg/liter of K2HPO4 was added, mixed, and added to 1.5 ml of serum in Kimax tubes (13 mm x 16 cm). Two ml of cold samples were centrifuged at 87(X) X g for 2 min. Rentinols were separated by chloroform were added, samples were gently mixed to avoid foaming, fol injecting 80 fil of supernatant onto a 25- x 4.6-mm ODS-2 high-performance lowed by centrifugation at 25(X) rpm for 10 min at 4Â°C.The supernatant was liquid chromatin column (Millipore-Whatman) and run in a mobile phase of quantitatively transferred into clean Kimax tubes (13 mm x 16 cm) by using a acetic acid/water/acetonitrilc (0.5/20.0/79.5) at 3.0 ml/min. Retinol and retinyl Pasteur pipete, followed by the addition of 2 ml of cold acetonitrile and acetate elute at 8 and 14 min, respectively, and were quantified by absorbance vigorous mixing by vortex. The mixture was centrifuged at 2500 rpm, the at 324 nm. Between-run error was estimated for the internal standard. The supernatant was saved, and the protein pellet was washed twice with 2 ml of standard deviation about the mean was 2.2% (n = 5), and similar variation was cold acetonitrile. The combined supernatants were evaporated under a stream observed for repeat runs of unknown serum samples. 1451

RESULTS swainsonine treatment. Decreases of 30% or more following treatment were observed in 4 patients, and this appeared to correlate with liver Treatment-related Effects and Patient Outcome. The character dysfunction (Table 2). An extended time course of serum retinol levels istics of the patients treated at 6 drug levels (50-550 jug/kg/day) are for patient VM/150 showed a decline of 50% following swainsonine summarized in Table 1. The major side effects observed with swain- treatment, and recovery of retinol levels by day 20 (Fig. 1). sonine treatment were edema and liver and pancreas dysfunction Assessment of disease response was not an end point of this study (Table 2). All patients showed treatment-related elevation of serum but patient AL/450 with head and neck cancer showed a 50-70% AST, while marginal increases in ALT were observed. Patients with reduction in measurable tumor mass for 6 weeks after a single 5-day normal serum AST levels on beginning treatment showed a rise of up to 4-fold during the 5 days of swainsonine infusion. The patients with treatment. Two patients reported an abrupt cessation of a significant most notable swainsonine-induced increases in AST/ALT were those preexisting cough during, and for 1 to 2 weeks after the swainsonine infusion. The latter patients were being treated at the 350-/j,g/kg/day who had elevated pretreatment levels. All but 1 of these patients (6 of level and both had lymphangitis carcinomatosis on chest 7) had known involvement of liver with malignancy. MDT (550 X-ray. fig/kg/day) was reached in patient B. P. who developed grade 4 Serum Drug Levels. As shown in Fig. 24, serum levels of swain elevation of bilirubin. This patient had significant involvement of her sonine were observed to increase rapidly during the first 8-12 h of liver with metastatic colon cancer and elevated transaminases and drug infusion, and approach steady state (Css) at 48-72 h. The serum hilirubin prior to receiving swainsonine. Five of 6 patients treated at MTD -1 developed < grade 3 toxicity which defined 450 /Â¿g/kg/day Css values ranged from 100 to 400 times greater than the KÂ¡for inhibition of Golgi a-mannosidase II (i.e., 20-30 ng/ml). Serum drug as the recommended starting dose for future clinical testing. levels were measured in VM/150 and VG/350 at intervals after dis A transient and asymptomatic rise in serum amylase was observed continuing swainsonine infusion and indicated a serum half-life of in 8 patients who began treatment with normal levels, and in 2 patients approximately 14 h (data not shown), consistent with the time re who started with elevated levels; 9 patients showed no change. These quired to reach Css (i.e., approximately 4 half-lives) (38). The CK changes in serum enzyme levels occurred at all doses with no apparent values for 16 courses of treatment plotted as a function of dosage in increase in severity over the dose range used in the trial. Fig. 28 shows a linear relationship but the line does not intersect the Edema was observed in 11 of 19 patients, and those patients who origin. The apparent nonlinear pharmacokinetics at doses of drug had multiple rounds of treatment showed reproducible levels of edema below 50 jug/kg/day suggests two serum compartments; one with slow (Table 2). Of the 2 patients (VM/150 and VG/350) who developed gross (4+) edema, both had pretreatment edema (2+, 1+, respec clearance (i.e., 0.5 ml/h/kg), and the other with a more rapid clearance tively). Edema was not consistently associated with severity of liver rate of 3.8 ml/h/kg). The mechanisms underlying the two clearance rates for swainsonine may be due to a swainsonine-binding molecule dysfunction, although all but 1 patient with known liver involvement with disease developed edema. Serum IL-6 and TNF-a levels were in serum which is saturated above 50 (ng/kg/day; and/or drug-induced measured in 10 patients. The two patients with 4+ edema on treatment activation of a clearance mechanism which enhances the clearance (VG/350 and VM/150), showed significant elevation of IL-6, 38- and rate at higher dosages (38). The latter may be explained for example 8-fold, respectively (Table 2). The mean IL-6 levels in patients before by conjugation of swainsonine, and will require further analysis of treatment was 12.0 Â±9.7 pg/ml (n = 10) and after 120 h of swain both swainsonine and its metabolites to address this question. sonine infusion it was 11.4 Â±6.2 pg/ml (n = 8). Serum TNF-a levels Inhibition of Golgi Oligosaccharide Processing a-Mannosidase. L-PHA lectin shows binding specificity for ÃŸl-6GlcNAc-branched were low and remained unchanged following swainsonine treatment. complex-type oligosaccharides (39) and can be used to measure the Other side effects noted in our patients included shortness of breath (n = 3; National Cancer Institute of Canada grade <2), lethargy (n â€” activity of swainsonine as an inhibitor of Golgi oligosaccharide pro 4; grade <2), nausea (n = 1; grade 1) and skin rash (n = 1; grade 1). cessing (i.e., a-mannosidase II). Glycoproteins synthesized in PBL during swainsonine treatment would be expected to bear hybrid-type Serum retinol levels were measured in 9 patients (10 courses) selected randomly at -24 h, 0 h, 96 h, and 120 h after beginning oligosaccharides rather than the L-PHA-reactive complex-type struc tures. Five days of swainsonine treatment reduced expression of L- PHA-reactive complex-type oligosaccharides on the surface of PBL Table 1 Prelrealmenl characteristics of patients enrolled on study lymphocytes by 2- to 7-fold based on mean fluorescent intensity (Fig. siteBreastDisease 3). A reciprocal increase in Con A binding to PBL was observed cancerNon-small consistent with the conversion of complex- to hybrid-type structures cell lung cancer (data not shown). The FACS analysis showed decreased L-PHA re Renal cellcancerColorectalUnknown activity in the total PBL population rather than evidence for multiple primarv cell populations, suggesting that a significant fraction of surface gly- Lymphoma 2 coproteins on circulating PBL were replaced during the 5 days of drug LeukemiaPancreas 222198:1136-73 cancerHead treatment. As a control, MDAY-D2 murine lymphoma cells were andneckn cultured in 1 /xg/ml of swainsonine for 48 h, to effect maximal (total)MalesrfemalesAge swainsonine-induced inhibition of processing (8), and these condi tions resulted in a 9-fold decrease in L-PHA binding to the cells (yr) (Fig.3). Range Urine Oligomannosides. In addition to Golgi a-mannosidase II, MedianPerformance 5177531 swainsonine also inhibits lysosomal a-mannosidases, and causes the statusECOG"0-123No. accumulation of oligomannosides in tissues and body fluids in animals (15, 40). FACE was used to quantify the reducing oligosaccharides in patient urine (Fig. 4). Urine oligosaccharides migrating in the FACE gels in the region of Glc, to Glc5 standards, accumulated rapidly over of coursesn42131 the first 72 h on swainsonine, and appeared to reach steady state by ' ECOG. Eastern Cooperative Oncology Group. 72 h (Fig. 5). Since serum swainsonine Css is attained in 48 h, and with 1452

Table 2 Summary of patient treatment, disease, edema, and changes in serum markers

Patient/doseTK/50LG/50WH/50VM/150EG/150SR/150MB/250LS/250AW/250GB/350MJ/350VG/350CV/450WL/450RC/450RB/450AL/450MS/450BP/550DiseaseBreastLeukemiaLymphomacBreast(grade)222(10224*11I)00(I0(I33(10114Â»10120001AST<35444883*908090*67*239b537234300b171"\&0b49W531123944127us*995b123"5962*to42636.?316"ALT<401953461363126136263117853337812181491417262636717441524121754Bilirubin<2010915103114151513206122220979111821151955"291217III9111167"'"Amylase<1155614239109%103563402251731109788142lit%17184â€”Â¡65ill97142"226*457739265Ã•55Grade(1,0,0,0)(1,0,0,0)(2,1,0,2)(1.0,3,0)(2,0,0,0)(1,0,0,0)(3,2,0,0)(1,0,0,3)(1,0,1,2)(0,0,0,2)(3,1,0,0)(2.0,1,0)(3,0,1,0)(1,0,0,1)(1,0,0,1)(1,0,0,0)(2,0,0,1)(1,0,0,0)(1,0,1,-)(2,0,0,1)(2,0,0,0)(4,3,3,0)(2,1,1,1)(1,0,0,2)(1,1,0,0)(1,0,0,0)(1,0,0,0)(1,0,0,3)(1,0,0,-)(3,1,4,1)IL-6NCâ€”â€”â€”NCâ€”8x

cColonLeukemiaUnknown17KidneyBreastBreastLungLung0'1*LymphomascPancreasPancreasColonHead/neckHead/neckColoncCourse1231212112112312121212111111121EdemaÃ®â€”â€”â€”NCâ€”â€”NCâ€”NCNCNCNCâ€”â€”38XTâ€”â€”â€”â€”â€”â€”â€”RA"â€”â€”â€”â€”â€”â€”1NCiNC_â€”â€”â€”â€”â€”â€”â€”â€”â€”iNCNCNCâ€”NCâ€”1

Upper limit for normals is indicated directly under the headings for AST, ALT (units/liter), bitirubin (ftmol/liter), and amylase (units/liter). The values are those measured on completion of 5 days of swainsonine infusion. Increases in serum AST, ALT,bilirubin, amylase after 5 days of swainsonine are indicated by italics. " RA, serum retinol levels; NC. no change; T, increase; i, decrease levels; â€”,not measured. h Abnormal pretreatment. '' Patients with liver mÃ©tastasespretreatment. d This patient died from progressive disease and acute respiratorydistress syndrome as suggested by autopsy. '' National Cancer Institute of Canada grade 4 bilirubin by day 3. Infusion stopped. MTD reached.

1.2 oligosaccharides by FACE gels similar, but not identical to that of swainsonine-treated patients (Fig. 4C). Analysis of PEL Cell Surface Markers. White cell counts showed that swainsonine was neither lympho- nor myelosuppressive 1.0 over the 5-day infusion. Swainsonine administered to mice has been shown to increase NK cell activity (23) and class II antigen la ex S pression in hematopoietic cell populations (30). Therefore, cell sur o E face antigens including CD3, CD4, CDS, HLA-DR, CD25, CD16, and aI 0.8 CD14 on the PEL of patients with nonhematological malignancies were examined in 7 patients (9 courses). Only HLA-DR showed a significant change with an increase of approximately 5-40% after 5 days of swainsonine treatment (P < 0.01) (Fig. 6). A similar increase 0.6 in the percentage of HLA-DR-positive cells was observed for PEL from healthy donors cultured for 3 days in the presence of swain-

0.4 O 2 4 6 8 1012141618 20 DISCUSSION Time (days) Fig. 1. Time course for serum retinol measurements in patient VM/150. , period In this first phase I trial of swainsonine, we have measured serum of swainsonine infusion; , minimum for serum retinol in healthy individuals. Css and i,/2, which allowed an estimate of serum clearance. The drug showed a relatively long serum half-life of 0.5 day. The serum Css an additional 24 h, a widespread effect of the drug also approaches values were 100 to 400 times greater than the X, for inhibition of steady state, it appears that the drug is rapidly distributed throughout Golgi a-mannosidase II, suggesting that the therapeutic dose may be the body. The swainsonine-induced oligosaccharides were susceptible significantly lower than that used in our study. In cell culture, the 50% to Jack bean a-mannosidase digestion, indicating that they were iso- inhibitory dose for Golgi oligosaccharide processing by swainsonine mers of Man2_5GlcNAc (Fig. 45). Urine oligosaccharides from pa is similar to the 50% inhibitory dose for a-mannosidases in vitro, tients with hereditary a-mannosidosis showed a pattern and level of suggesting that the drug has excellent cell membrane permeability 1453

(41). Golgi a-mannosidase II was markedly inhibited in PBL as in ferred by the loss of L-PHA-binding complex-type oligosaccharides after 5 days of treatment. Tissue lysosomal a-mannosidases were also inhibited by swainsonine, and saturation of this drug-related effect >Â« 8 was observed within 24 h of reaching steady-state levels of drug in serum. Based on these measurements, dosages of 150 and 450 /Â¿g/ aCB â€¢3 kg/day showed indistinguishable levels of inhibition for Golgi and a lysosomal a-mannosidases, which suggests that these biological ef fects are saturated above 150 ;u.g/kg/day. This is supported by the observation that the amount of urine oligomannosides after 5 days of swainsonine treatment was similar to that observed in patients with the genetic disorder a-mannosidosis. Although swainsonine was well tolerated in our patient group, definite treatment-related side effects included abnormal liver func tion tests, edema, and elevation of pancreatic amylase. These effects did not appear to correlate with one another or with drug dosage. Hepatic toxicity was reflected by transaminase elevation, and the time PrÃ© Post course suggests acute nonspecific hepatocellular damage. Significant Fig. 3. L-PHA lectin binding to PBL of patients pre- and postswainsonine treatment. Two samples of PBL. at -24 h and at initiation of swainsonine treatment, were compared elevation of liver enzymes only occurred in patients with known to samples taken at 120 h after treatment. Measurements for L-PHA binding were taken hepatic tumor involvement. In preclinical tests of swainsonine in two on 8 courses of treatment; WL/450, MSI/450, RC/450, RB/450, EG2/150, AL/450, SR/150, MS2/150. For comparison, MDAY-D2 murine lymphoma cells (O); were cultured in vitro absence (pre) and presence of 1 /ig/ml of swainsonine for 48 h (post).

G5-.

G4-.

Day:

24 48 72 96 120 144 B Time (hours)

10 - B

E oÃ

Fig. 4. A, fluorescent image of FACE gel showing separation of oligosaccharides from urine of a patient treated with 150 jig/kg/day; EG1/150. B, Lane 1 is 5-day urine from a swainsonine-treated patient; Lane 2 is the same urine digested overnight with 50 milliunits of Jack bean a-mannosidase. (C). Lane I is 5-day urine from a swainsonine-trealed patient: Lane 2 is urine from an a-mannosidosis patient.

Rhesus monkeys, a 5-fold increase in AST levels was observed when O 100 200 300 400 500 600 the drug was given p.o. at 8 mg/kg/day over 36 days.5 This is similar doso (ug/kg/day) to the rise in AST and ALT observed in our patients treated with Fig. 2. A, representative time course of swainsonine accumulation in the serum of swainsonine, but at doses approximately 16 times greater than that patients receiving â€¢,50; T, 150; O, 250; and V, 450 jug/kg/day of swainsonine. B, mean used in our trial. MTD was defined in a patient with significant serum Câ€žsvalueswere calculated by averaging serum measurements at 72, 96. 120 h; SD. <15%. The C\s values were plotted for 16 courses of swainsonine treatment. Clearance pretreatment hepatic dysfunction and it is possible that higher levels of (i.e., CL = dose/Css) was significantly different for 8 patients receiving 50-250 |tg/kg/day compared to the 8 patients who received 350-550 (ig/kg/day (t test, P < 0.0002). which suggests nonlinear pharmacokinetics (38). 1J. J. Lipman, Vanderbilt University, personal communication. 1454

swainsonine, the pyrrolizidine alkaloids have a double bond at 1,2 and 10 are bioactivated to pyrrolic dehydroalkaloids which are mutagenic and g hepatotoxic (43). The pyrrolizidine alkaloids are found in tansy rag wort (Senecio jacobaea), and their consumption by rats and chickens a results in hepatotoxicity associated with depletion of liver retinol stores (44). In chickens fed a diet supplemented with tansy ragwort, the liver histopathology could be prevented by a vitamin A supple ment, suggesting that its depletion was a significant factor in hepato toxicity (44). Four of 9 swainsonine-treated patients examined showed evidence of drug-related depletion of serum retinol. Therefore in subsequent clinical studies of swainsonine, analysis of serum retinol o 3 en levels and hepatotoxicity must be examined and consideration be O .? 2 given to intervention with vitamin A supplements. Studies in mice suggest that swainsonine therapeutic effects are related to inhibition of Golgi a-mannosidase II (19). Although re versible on withdrawal of drug, the accumulation of oligomanno- -10123456 sides due to inhibition of lysosomal a-mannosidases is a side effect, Time of treatment (days) which on long-term administration of swainsonine, may become a Fig. 5. Accumulation of oligosaccharide in urine of swainsonine-trcatcd patients. Data significant problem. The number of major oligomannoside species are derived from densitometry reading of FACE gels where the densities of hands which observed in the urine of swainsonine-treated patients was greater migrate with the Glc4 and Glc5 standards as shown in the boxed area of Fig. 4A have been summed and expressed as a ratio of urine: creatinine levels. Patients were WL/450 (D), than that of an a-mannosidosis patient (Fig. 4C). a-Mannosidosis MSI/450 (A), RC/450 (â€¢).RB/450 (â€¢),EG1/150 (Y). EG2/150 (O), VM/150 (V). The patients are deficient in lysosomal a 1-3 mannosidase, and for rea values were normalized to pretreatment urine and expressed as fold increase. The creati nine levels in serum were 3.9 Â±2.0 mg/ml (Â«= 28). and swainsonine therapy had no sons which are not entirely clear, accumulate three major storage apparent effect on creatinine levels. products in tissues and urine (i.e., Manal-3ManÃŸl-4GlcNAc and Manal-2Manal-3ManÃŸl-4GlcNAc, Manal-2Manal-2Manal- 3ManÃŸl-4GlcNAc) (45). Swainsonine inhibits both al-3 mannosi dase as well as the recently described a 1-6 mannosidase (39, 46), and therefore results in the accumulation of two major storage prod ucts; Manal-6[Manal-3]Manal-6[Manal-3]ManÃŸl-4GlcNAc and Manal-6[Manal-3]ManÃŸl-4GlcNAc in fibroblasts (46). The more heterogeneous mixture of oligomannosides in the urine of swain sonine-treated patients probably reflects incomplete inhibition of the lysosomal a-mannosidases, which results in the accumulation of Manal-3ManÃŸl-4GlcNAc,Manal-6[Manal-3]ManÃŸl-4GlcNAc, as well as isomers of Man4.5GlcNAc (see Fig. 44 ). K a 10 - HLA-DR, a class II antigen found on monocytes, activated T-cells, I and NK cells, was elevated in PBL of swainsonine-treated patients, 5 i similar to that observed for PBLs from healthy subjects cultured in the presence of swainsonine for 3 days. In mice, thioglycollate-elicited peritoneal macrophages were shown to be activated by culturing the cells in the presence of swainsonine or by injecting swainsonine into the peritoneal cavity (30, 31). The macrophages showed increased Pre Post tumoricidal activity, IL-1 production, induction of protein kinase C Fig. 6. HLA-DR levels on PBL measured before (pre) and after 5 days of swainsonine activity, and increased cell surface class II histocompatibility antigen treatment (post). For each patient, two pretreatmcnt blood samples were analyzed, and la. In these studies, stimulation of tumoricidal activity by swainsonine either 1 or 2 samples on day 5 following completion of swainsonine infusion. â€¢,PBL from patients EG1/150; EG2/150; MSI/450; MS2/450; RC/450; RB/450; WL/450; AL/450, and was found to be comparable to that of other activating agents includ LS/250. Applying the signed rank sum test, the increase in HLA-DR is significant at P < ing Corynebacterium parvum, lipopolysaccharide and IFN-7. 0.01. In comparison, analysis of this group of patient for CDI6 showed a tendency to increase, P = 0.15; and no significant change in CD3, CD4, and CD25, P > 0.5. O, PBL Swainsonine is a potent immune modulator in mice, stimulating from healthy volunteers cultured for 3 days either without (pre) or with 10 ^.g/ml of lymphocyte proliferation (29), activating natural antitumor immunity swainsonine (post). (23, 32), and enhancing T-cell stimulation by antigen (47). In mice treated with chemotherapeutic drugs, swainsonine has been shown to drug would be well tolerated in patients without liver mÃ©tastasesor enhance bone marrow cellularity (27) (reviewed in Ref. 26). The basis liver dysfunction secondary to other causes. It is worth noting that of immune cell activation and bone marrow proliferation may be there was no evidence of neurotoxicity in the two rhesus monkeys or related to the observation that some cytokines and growth factors have in the 19 patients in our trial, an effect which has been observed in carbohydrate-binding activities (48). IL-1, IL-2, and TNF bind to grazing animals that consume "loco weed," a natural source of swain oligomannose structures (49), which are made available on the cell sonine and several other alkaloids (13, 42). surface when cells are grown in the presence of swainsonine (i.e., the Rats fed a diet supplemented with swainsonine have previously Manal-6(Manal-3)Manal-6ManÃŸ portion of the hybrid-type struc been shown to manifest reversible liver vacuolization but without tures). In this regard, human PBL cultured with swainsonine enhances oligosaccharide accumulation or hepatocyte death (33). Swainsonine LAK cell killing of human colon carcinoma cells (32). Similar results does not appear to be mutagenic in cell culture and does not cause were obtained with 1-deoxymannojirimycin, but castanospermine and acute liver cell damage in animals, in sharp contrast to the pyrroliz- 1-deoxynojirimycin had no effect. Antibodies to IL-2 abolished idine alkaloids, a structurally related class of compounds. Unlike swainsonine-induced enhancement of LAK cell killing but lympho- 1455

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1994 American Association for Cancer Research. SWAINSONINE IN ADVANCED MALIGNANCIES cytes showed no increase in IL-2 production or receptor number. REFERENCES These observations are consistent with the hypothesis that a-manno- Yamashila, K., Ohkura, T., Tachibana, Y., Takasaki, S., and Kobata, A. Comparative sidase inhibitors increase the sensitivity of LAK cells to IL-2 activa study of the oligosaccharides released from baby hamster kidney cells and tion (32). Infiltrating lymphocytes and monocytes in tumors are their polyoma transformant by hydrazinolysis. J. Biol. Chem., 259: 10834-10840, 1984. largely inactive, but can be activated in vitro by IL-2 and other Pierce, M., and Arango, J. Rous sarcoma virus-transformed baby hamster kidney cells cytokines (50), suggesting that swainsonine might serve to sensitize express higher levels of asparagine-linked Iri- and tetraantennary glycopeptides con LAK cells, NK cells, and macrophages to local cytokines in the tumor. taining [GlcNAc-ÃŸ(l,6)Man-a(l,6)Man] and poly-W-acetyllactosamine sequences than baby hamster kidney cells. J. Biol. Chem., 261: 10772-10777, 1986. The edema observed in swainsonine-treated patients was not generally Dennis, J. W., Kosh, K., Bryce, D-M., and Breitman. M. Oncogenes conferring associated with increased serum cytokine levels (i.e., IL-6 and TNF), metastatic potential induce increased branching of Asn-linked oligosaccharides in rat2 but may result from a sensitization of vascular endothelium to endo fibroblasts. Oncogene. 4: 853-860, 1989. Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L., and Kerbel, R. S. 01-6 genous cytokines, which may also be higher at sites of bulky tumor branching of Asn-linked oligosaccharides is directly associated with metastasis. burden. The potential relationship between treatment-related severe Science (Washington DC), 236: 582-585, 1987. edema and elevated IL-6 observed in patients will require further Dennis, J. W., and Laferte, S. Oncodevelopmental expression of -GlcNAc ÃŸl-6Man al-6Man ÃŸl-6branching of Asn-linked oligosaccharides in human breast carcinomas. study. Cancer Res., 49: 945-950, 1989. The shortness of breath noted in 3 patients was mild and not dose Fernandes. B., Sagman, U., Auger, M., Demetrio, M., and Dennis, J. W. ÃŸl-6 dependent, and like the peripheral edema, may represent "capillary branched oligosaccharides as a marker of tumor progression in human breast and leak" in the pulmonary vasculature. One patient (VG/350), developed colon neoplasia. Cancer Res., 51: 718-723, 1991. Dennis, J. W. Different metastatic phenotypes in two genetic classes of wheat germ significant edema and liver dysfunction in association with respiratory agglutinin-resistant tumor cell mutants. Cancer Res., 46: 4594-4600, 1986. Dennis, J. W. Effects of swainsonine and polyinosinic-polycytidylic acid on murine failure. Postmortem findings indicated significant tumor within the tumor cell growth and metastasis. Cancer Res., 46: 5131-5136, 1986. lungs and liver, as well as bilateral pneumonia. It was thought at the Humphries, M. J., Matsumoto, K., White, S. L., and Olden, K. Oligosaccharide time that the patient died of progressive disease, but in retrospect a modification by swainsonine treatment inhibits pulmonary colonization by BI6-F10 murine melanoma cells. Proc. Nati. Acad. Sci. USA, 83: 1752-1756, 1986. capillary leak resulting in acute respiratory distress syndrome may Pulverer, G., Beuth, J., Ko, H. L., Yassin, A.. Ohshima. Y., Roszkowski. K., and have been caused by swainsonine. This patient has therefore been Uhlenbruck, G. Glycoprotein modifications of sarcoma L-l tumor cells by tunica- recorded as a possible treatment-related death. As suggested above, mycin, swainsonine, bromoconduritol or 1-deoxynorjirimycin treatment inhibits their metastatic lung colonization in Balb/c-mice. J. Cancer Res. Clin. Oncol., 114: 217- severe liver involvement with tumor may have provoked local cyto 220, 1988. kine release in the lung, and sensitization by swainsonine may have Humphries. M. J., Matsumoto, K., White. S. L., and Olden. K. Inhibition of experi mental metastasis by castanospermine in mice: blockage of two distinct stages of provoked the syndrome. tumor colonization by Oligosaccharide processing inhibitors. Cancer Res., 46: 5215- Swainsonine appears to have several anticancer actions which may 5222, 1986. vary in importance for different types of tumors. These include: (a) Colegate, S. M., Huxtable, C. R., and Dorling, P. R. A spectroscopic investigation of swainsonine: an alpha-mannosidase inhibitor isolated from Swainsona canescens. inhibition of tumor cell metastasis and invasion by direct action on the Aust. J. Chem., 32: 2257-2264, 1979. tumor cells as an oligosaccharide-processing inhibitor; (b) inhibition Molyneux, R. J., and James, L. F. Loco intoxication: indolizidine alkaloid of spotted of solid tumor growth, at least partially by direct action of drug on the locoweed. Science (Washington DC), 216: 190-191, 1981. Schneider. M. J., Ungemach. F. S., Broquist, H. P., and Harris, T. M. Tetrahedron Lett., tumor cells; and (c) augmentation of immune function including 39: 29-31, 1983. LAK, NK cell, and macrophage activities. The relative importance of Tulsiani. D. R., Harris, T. M.. and Touster, O. Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi mannosidase II. J. Biol. Chem., 257: the action of swainsonine on tumor cells and on host immunity is 7936-7939, 1982. unclear. If stimulation of host immunity is the target, it may be best to Dorling, P. R., Huxtable, C. R., and Colegate, S. M. Inhibition of lysosomal alpha- use modest doses of swainsonine on a longer interval as per a study in mannosidase by swainsonine, an indolizidine alkaloid isolated from Swainsona canescens. Biochem. J., 191: 649-651, 1980. mice by Humphries et al. (23, 24). This strategy could be used in DeSantis, R., Sanier, U. V., and Click, M. C. NIH 3T3 cells transfected with human combination with other immunostimulants such as IL-2 and IFN, tumor DNA lose the transformed phenotype when treated with swainsonine. Biochem. which have been shown to have additive antitumor activity in mice Biophys. Res. Commun., Â¡42:348-353, 1987. Dennis, J. W., Koch, K., Yousefi, S., and VanderElst, I. Growth inhibition of human (51). Ionizing radiation has been shown to induce cytokine production melanoma tumor xenografts in athymic nude mice by swainsonine. Cancer Res., 50: by tumor cells including TNF-a, which may contribute to radiation 1867-1872, 1990. lethality (52). Therefore, swainsonine might be used to sensitize tu VanderElst, L, and Dennis, J. W. A'-Linked Oligosaccharide processing and autocrine- stimulation of tumor cell proliferation. Exp. Cell Res., 192: 612-613, 1991. mors to irradiation therapy, and at the same time provide bone marrow Cornil, L, Kerbel, R. S., and Dennis, J. W. Tumor cell surface ÃŸl-4linked galactose protection. binds to lectin(s) on microvascular endothelial cells and contributes to organ coloni In summary, this first phase I trial of swainsonine administered i.v. zation. J. Cell Biol., 7/7: 773-782, 1990. Yagel, S., Feinmesser, R., Waghorne, C., Lala, P. K., Breitman, M. L., and Dennis, J. at 50 to 550 ftg/kg/day shows that the drug is well tolerated in cancer W. Evidence that ÃŸl-6branched Asn-linked oligosaccharides on metastatic tumor patients, particularly when the liver is not compromised by tumor cells facilitate invasion of basement membranes. Int. J. Cancer, 44: 685-690, 1989. Seftor, R. E. B., Seftor, E. A., Grimes, W. J., Liotta, L. A., Stetler-Stevenson, W. G., involvement. The dosages used in this study appear to be saturating 22. Welch, D. R., and Hendrix, M. J. C. Human melanoma cell invasion is inhibited in for 2 drug-related effects, inhibition of Golgi a-mannosidase II in vitro by swainsonine and deoxymannojirimycin with a concomitant decrease in col- PBL, and tissue lysosomal a-mannosidases. Increased levels of HLA- lagenase IV expression. Melanoma Res., I: 43-54, 1991. DR-positive lymphocytes, objective clinical response in 1 patient, and 23. Humphries, M. J., Matsumoto, K., White. S. L., Molyneux, R. J., and Olden. K. Augmentation of murine natural killer cell activity by swainsonine, a new antimeta- symptomatic improvement in another 2, suggest that swainsonine may static immunomodulalor. Cancer Res., 48: 1410-1415, 1988. have therapeutic activity in cancer patients. Further studies are under 24. Humphries, M. J., Matsumoto, K., White, S. L., Molyneux, R. J., and Olden, K. An assessment of the effects of swainsonine on survival of mice injected with B16-F10 way to assess bioavailability and toxicity of swainsonine administered melanoma cells. Clin. Exp. Metastasis, 8: 89-102, 1990. p.o. to cancer patients. 25. Kino, T., Inamura, N., Nakahara, K., Kiyolo, S., Goto, T., Terano, H., Kohsaka, M., Oaki, H.. and Imanaka, H. Effect of swainsonine on mouse immunodeficient system and experimental murine tumor. Journal Antibiot. (Tokyo), 38: 936-940, 1985. ACKNOWLEDGMENTS 26. Olden, K., Breton, P., Grzegorzewski, K., Yasuda, Y, Gause, B. L., Oredipe, O. A., Newton, S. A., and White, S. L. The potential importance of swainsonine in therapy We thank Dr. Frances Shepherd and Dr. Michael Crump for overseeing for cancers and immunology. Pharmacol. Ther., 50: 285-290, 1991. some patients during swainsonine infusion; Virgilio Deleon and Karen Paul for 27. Oredipe, O. A., White, S. L., Grzegorzewski, K., Gause, B. L., Cha, J. K., Miles, V. A., and Olden, K. Protective effects of swainsonine on murine survival and bone research assistance; Dr. Chris Starr, GLYCO, Inc., for FACE analysis of urine marrow proliferation during cytotoxic chemotherapy. J. Nati. Cancer Inst., 83: 1149â€” oligosaccharides and helpful comments; and Cathy Bedlington and Zofia 1156, 1991. Kryzek for secretarial assistance. 28. White, S. L., Nagai, T, Akiyama, S. K., Reeves, E. J., Grzegorzewski, K., and Olden, 1456

K. Swainsonine stimulation of the proliferation and colony forming activity of murine 41. Dennis. J. W.. White. S. L.. Freer. A. M., and Dime, D. Carbonoyloxy analogues of hone marrow. Cancer Commun.. 3: 83-91, 1991. the anti-melastatic drug swainsonine: activation in tumor cells by esterases. Biochem. 29. While, S. L., Schweitzer, K., Humphries, M. J., and Olden, K. Stimulation of DNA Pharmacol., 46: 1459-1466. 1993. synthesis in murine lymphocytes hy the drug swainsonine: immunomodulatory prop 42. Tulsiani. D. R.. Broquist. H. P., James. L. F., and Touster, O. The similar effects of erties. Biochem. Biophys. Res. Commun., ISO: 615-625. 19X8. swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig 30. Grzegorzewski, K., Newton, S. A.. Akiyama, S. K., Sharrow. S.. Olden, K., and indicate that the alkaloid is the principal toxin responsible for the induction of White, S. L. Induction of macrophage tumoricidal activity, major histocompatihility locoism. Arch. Biochem. Biophys., 232: 76-85, 1984. complex class II antigen (lak) expression, and interleukin-l production hy swainso 43. Frei, Hâ€žLÃ¼thy,J.,Brauchli, J., Zweifel, U., WÃ¼rgler,F.E., and Schlauer. C. Structure/ nine. Cancer Commun.. /: 373-379, 1989. activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids 31. Breton, P., Asseffa, A.. Grzegorzewski, K., Akiyama. S. K., White. S. L., Cha. J. K., assayed for the induction of somatic mutation and recombination in wing cells of and Olden. K. Swainsonine modulation of protein kinasc C activity in murine peri Drosophi/u mclanogasier. Chem.-Biol. Interact., 83: 1-22, 1992. toneal macrophages. Cancer Commun., 2: 333-338. 1990. 44. Huan, J.. Cheeke, P. R., Lowry, R. R., Nakaue, H. S., Snyder, S. P., and Whanger. 32. Vagita. M., and Saksela. E. Swainsonine. an inhihitor of glycoprotein processing, P. D. Dietary pyrrolizidine (Senecio) alkaloids and tissue distribution of copper and enhances cytotoxicity of large granuul lymphocytes. Scand. J. Immunol., 31: 275- vitamin A in broiler chickens. Toxicol. Lett. (Amst.), 62: 139-153, 1992. 282, 1990. 45. AI Daher, S.. De Gasperi. R., Daniel. P., Hall, N., Warrern, C. D., and Winchester, B. 33. Novikoff, P. M., Touster, O., Novikoff, A. B., and Tulsiani, D. P. Effects of swain The substrate-specificity of human lysosomal alpha-i>-mannosidase in relation to sonine on rat liver and kidney: hiochemical and morphological studies. J. Cell Biol., genetic alpha-mannosidosis. Biochem. J., 277: 743-751. 1991. 101: 339-349. 1985. 46. Tulsiani, D. R.. and Touster, O. Substrate specificities of rat kidney lysosomal and 34. Pritchard, D. H., Huxtable, C. R., Dorling. P. R.. and Colegate. S. M. The effect of cytosolic a-D-mannosidases and effects of swainsonine suggest a role of the cytosolic swainsonine on the growth rate of young rats. In: L. F. 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Paul E. Goss, Jose Baptiste, Berny Fernandes, et al.

Cancer Res 1994;54:1450-1457.

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