Phase lB Clinical Trial of the Oligosaccharide Processing Inhibitor Swainsonine in ... · edema....

Vol. 3. 1077-If)86. Jim/v /997 Clinical Cancer Research 1077

Phase lB Clinical Trial of the Oligosaccharide Processing Inhibitor

Swainsonine in Patients with Advanced Malignancies1

Paul E. � Caroline L. Reid, Dennis Bailey,

and James W Dennis4

The Toronto Hospital. Department of Medical Oncology. MLW

2-022. 200 Elizabeth Street. Toronto. Ontario. M5G 2C4 Canada

P. E. G.. C. L. R.. D. B.I. and Samuel Lunenfeld Research Institute.Mount Sinai Hospital. 600 University Avenue. Toronto, Ontario.

M5G IXS Canada Ii.W. D.I

ABSTRACTThe indolizidine alkaloid swainsonine, a potent inhibi-

tor of Golgi ot-mannosidase II, has been shown to reduce

tumor cell metastasis, enhance cellular immune responses,

and reduce solid tumor growth in mice. In our previous

Phase I study, swainsonine administered by 5-day continu-

ous infusion inhibited 1.-phytohemagglutinin-reactive

N-linked oligosaccharide expression on peripheral blood

lymphocytes. Significant toxicities included edema and ele-

vated serum aspartate aminotransferase (AST). One patient

with head and neck cancer had objective (>50%) tumor

remission. Two patients showed symptomatic improvement.

The objectives of this Phase lB trial were to examine the

pharmacokinetics, toxicities, and biochemical effects of bi-

weekly oral swainsonine at escalating dose levels (50-600

�Lg/kg) in 16 patients with advanced malignancies and 2

HIV-positive patients unsuitable for conventional therapy.

Eastern Cooperative Oncology Group performance status

was S2. The maximum tolerated dose was defined as 300

pig/kg/day due primarily to serum AST abnormalities and

dyspnea. Other adverse events present in >20% of patients

included increase in serum AST (all patients), fatigue (ii

9), anorexia (it = 6), dyspnea (n 6), and abdominal pain

(n = 4). Inhibition of Golgi a-mannosidase II occurred in a

dose-dependent manner. Examination of immunological pa-

rameters revealed a transient decrease in CD25� peripheral

blood lymphocytes and, in seven of eight patients, an in-

crease in CD4�:CD8� ratios at 2 weeks. Serum drug levels

peaked 3-4 h following a single oral dose in most patients

and were proportional to dose at levels � 150 pg/kg. We

conclude that oral swainsonine is tolerated by chronic inter-

Received 5/22/96: revised 3/1 2/97: accepted 4/2/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must thereftre be hereby marked

advertisement in accordance with I 8 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported by grants from the National Cancer Institute

of Canada Breast Cancer Research Initiative (to P. E. G. and J. W. D.).

I. W. D. is a Senior Research Scientist of the National Cancer Institute

of Canada.

2 To whom requests for reprints should he addressed.

3 Consultant to GlycoDesign. Inc.

4 Co-address: GlycoDesign. Inc.. 480 University Avenue. Suite 900,

Toronto. Ontario, M5G 1V2 Canada.

mittent administration at doses up to 150 �igIkg/day. Ad-

verse events considered drug related were similar to those

observed in the infusional study but with fatigue and neu-

rological effects also noted. Investigations of alternative dos-

ing schedules with low starting doses are suggested for

further clinical testing.

INTRODUCTION

Cell surface carbohydrate structures participate in cell-cell

and cell-substratum interactions affecting lymphocyte traffick-

ing, immutie cell stimulation and function, embryogenesis. and

cancer metastasis ( I-3. Structural diversity of carbohydrates

found on secreted and transmembrane glycoproteins are a result

of tissue-specific regulation of Golgi enzymes required in their

biosynthesis (Refs. 4-6; Fig. I ). Cancer cells commonly show

increased GlcNAc5-transferase V activity and the resulting

�3l-6 G1cNAc-branched N-linked carbohydrate structures (7-

10). We have shown that the degree of �l-6GIcNAc-hranching

in rodent tumor models ( I I. 12) and in human breast and colon

carcinomas (13. 14) correlates with disease progression. Fur-

thermore, inhibitors of the N-linked carbohydrate processing

pathway have been shown to attenuate both metastasis and

tumor growth in animal models (15-18).

Swainsonine, or 8pi3-indolizidine-Ia.2a,8b-triol. is an in-

dolizidine alkaloid first isolated from the Australian plant

Sttain.soiia ((1FU’S((’flS ( 19), and later from North American

plants of the genera A.stragalus and Oxvtropis ( 20). Swainsonine

inhibits Golgi a-mannosidase II and consequently the carbohy-

drate processing pathway prior to the initiation of the �l-6

GlcNAc-Iinked branch (Fig. I . Tumor cells cultured in the

presence of swainsonine show enhanced substratum adhesion

and loss of invasiveness into extracellular matrix, as well as loss

of organ colonization potential when injected iv. into mice ( 16,

21, 22). Given to mice p.o. (15, 23-25), by i.p. injection (26),

and by systemic infusion (27), swainsonine has been shown to

inhibit solid tumor growth of murine lymphoma as well as

human melanoma and breast and colon carcinoma xenografIs. In

addition to the effects on tumor cell invasion. swainsonine

alleviates tumor-induced and chemically induced immune sup-

pression (26, 28. 29) and stimulates both natural killer cell (24)

and niacrophage (30. 3 1 ) activity in mice. The addition of

swainsonine to human lymphocyte cultures has been shown to

augment lymphocyte-activated killer cell-induced killing of hu-

man colon carcinoma cells (32). These observations strongly

suggest that swainsonine may be useful for the treatment of

human malignancy.

S The abbreviations used are: GlcNAc. N-acetylglucosamine: MTD.

maximally tolerated dose: DLT. dose-limiting toxicity: AST. aspartate

aminotransferase: PHA. phytohemagglutinin: PBL. peripheral blood

lymphocyte: FCM. flow cytometry FITC. fluorescein isothiocyanate.

Research. on August 4, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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hybrid-typeA

Binds L-PHA

complex-typeA A Ai I I I I

�3�G �3�MI �Y�1�a��MIl t? iii U ip,i � rv�Y

rr-0i-� i

1078 Phase lB Study of Swainsonine

Gic A

GIcNAc �

Man 0Gal #{149}SA G

Two Phase I B studies of swainsonine in patients with

advanced malignancies have now been completed. In our first

study (33). swainsonine was administered to 19 patients at

repeated. 28-day intervals by continuous iv. infusion over 5

days in escalating doses from 50-550 pgfkg/day. The MID and

the reconiiiiended starting dose (MID-I level) were 550 and

450 pg/kg/day. respectively. Increases in serum AST levels

were dose limiting. Other adverse events included dyspnea.

edema. fatigue. anorexia, mild alanine aminotransferase eleva-

Lion, a rise iii serum arnylase. and decreased serum retinol levels.

One patient died from an acute respiratory distress syndrome

that was possibly treatnient related, although postmortem find-

ings indicated significant tumor within the lungs and liver. as

well as bilateral pneumonia. One patient with head and neck

cancer showed >50% shrinkage of tumor mass. A marked

decrease in i.-PHA binding to PBLs was detected after the 5-day

course of treatnient. consistent with inhibition of the target

enzyme Golgi a-mannosidase II. Oligoniannosides accumulated

iii patient urine, reflecting inhibition of tissue lysosomal a-man-

nosidases, and reached steady state at 3 days. only I day after

serum drug steady state.

Here we describe our second clinical study that examined

the use of swainsonine given by oral administration in twice

weekly doses of 50-600 p.g/kg. In animal studies. inhibition of

solid tumor growth and immune stimulation has been demon-

strated when swainsonine was given p.o. either by chronic or

intermittent schedules (24). In the present Phase lB study of oral

swainsomne, we have measured qualitative and quantitative

toxicities, serum drug levels, and markers of a-mannosidase

inhibition and lymphocyte distribution.

PATIENTS AND METHODS

Eighteen patients were enrolled in the chronic oral study

between July 1993 and February 1994. The study was approved

by the University of Toronto and The Toronto Hospital Ethics

Coiiiiiiittee. Patients � I 8 years with a life expectancy of at least

3 months, Eastern Cooperative Oncology Group perftwmance

status 0-2, and histologically confirmed diagnosis of metastatic

cancer refractory to standard effective therapy. or for which no

conventional therapy exists, were eligible for enrollment on

Fig. I The Golgi N-linked carbohydrate

processing pathway. aG, a-glucosidase: aM!,

a-maniiosidase I: aM!!, a-mannosidase II: TI.

TI!. TI� and TV. the respective GlcNAc-Ts:

SW, swainsonine: Mami. mannose: Gal. galac-tose: SA. sialic acid. The [3I-6GIcNAc-

branched complex-type structure requiring

GIcNAc-TV is shown on the rig/it with the

L-PHA-binding site boxed. GIcNAc-TV achy-

it)’ is up-regulated in cancer cells and in acti-

sated T lymphocytes.

study. HIV-positive patients unsuitable for conventional therapy

were also eligible. All patients were required to have completed

pre-study cheniotherapy or radiotherapy at least 3 weeks prior to

enrollment and have signed an informed consent. Exclusion

criteria included inadequate organ function (hepatic: bilirubin

>20 jimol/liter or AST �2 times normal: renal: creatinine

>130 or creatinine clearance �1 mI/s). central nervous sys-

teni metastases, or prior exposure to experimental anticancer

agents. Screening prior to enrollment included: signed in-

formed consent: history and physical exam: assessment of

Eastern Cooperative Oncology Group performance status:

chest X-ray: routine hematology. biochemistry. urinalysis.

and electrocardiogram: blood and urine collection for base-

line PBL marker analysis by FCM: plasma drug levels: and

urine oligomannosides. Baseline symptoms were recorded.

During the chronic oral dose schedule patients were exam-

med every 2 weeks, at which time any observed toxicities were

recorded and physical examinations were performed. Routine

hematology. biochemistry. plasma drug level. and urine oligo-

mannoside analysis were done at each visit. FCM analysis was

conducted before treatment initiation and at 2 and 4 weeks of

biweekly swainsonine treatment. All samples were collected

prior to swainsonine administration that day. Some patients did

not return for follow-up visits at exact 2-week intervals or did

not provide blood and/or urine samples. Only those patients with

complete blood sample sets (baseline and 2 and 4 weeks on

biweekly swainsonine treatments) were included in our FCM

data analysis. Blood and urine was not collected from HIV-

positive individuals. Chest X-rays and EKGs were repeated as

determined appropriate by the investigator.

Swainsonine Administration. Swainsonine was synthe-

sized by Toronto Research Chemicals. Purity was confirmed by

proton nuclear magnetic resonance spectroscopic analysis to be

greater than 98%. Appropriate concentrations of swainsonine

were prepared by the Pharmacy Department of the Toronto

Hospital by reconstituting the drug in 30 ml of sterile water in

30-mI/unit dose polypropylene vials. Reconstituted swainsonine

is a colorless and tasteless preparation. Chronic oral scheduling

consisted of twice weekly doses (Mondays and Thursdays)



Clinical Cancer Research 1079

administered before the evening meal. The chronic oral dose

levels were 50, 150. 300, and 600 p.g/kg on each treatment day.

Pharmacokinetics. Swainsonine was prepared for single

oral bolus administration in concentrations of 50. 150, 250, 500,

700, and 1000 p.gfkg. Blood samples were drawn from a saline

lock inserted into the patient’s arm vein at t = 0. 0.33, 0.66, 1.0,

1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, and 6.0 h following swainsonine

administration. Serum swainsonine levels were fit to multiex-

ponential equations by nonlinear regression. The relatively short

sampling interval allowed only two pharmacokinetic parame-

ters, maximum swainsonine concentration (C,,,.�) and time to

maximum (T0,,�), to be calculated.

Dose Escalation. Three patients were treated at each

dose level. Intrapatient dose escalation was not allowed. Dose

escalation occurred until the MID was reached. MID was

defined as that dose level at which at least two of three patients

experienced DLI. Once all three patients in a cohort had been

followed for 1 month (from their first dose of swainsonine) and

evidence of DLI was seen either in one or fewer of these

patients. new patients were treated at the next higher dose level.

DLI was defined as any grade 3 or 4 nonhematological toxicity

according to standard National Cancer Institute of Canada Lox-

icity criteria (used with the permission of the National Cancer

Institute of Canada). When MID was defined, three additional

patients were treated one dose level lower to ensure that MID

had not been exceeded.

Serum Drug Levels. The method used for extraction,

acetylation, and measurement of serum swainsonine has been

slightly modified from our protocol published previously (34).

The dry samples were acetylated by the addition of I 8 mg of

4-dirnethylaminopyridine. 18 mg of sodium acetate, I ml of

acetonitrile, and 500 p.1 of acetic anhydride. The sealed reaction

tubes were heated overnight in a 70#{176}Cwater bath. The mixture

of reaction products were partitioned by adding I ml of chlo-

roform, I ml of water, and mixing. The lower chloroform layer

was transferred by Pasteur pipette to a clean tube. Two further

additions of I ml of chloroform were made to the mixture of

products and mixed; then the lower chloroform layer was pooled

for each sample. The chloroform solution was washed with 1 ml

of water to further remove polar products and unreacted re-

agents from the solution. The lower chloroform layer was

passed through a solid phase extraction cartridge of 500 mg of

basic alumina to clean the sample; then it was concentrated

under nitrogen at 60#{176}C.The samples were quantitatively trans-

ferred to GC vials with micro inserts, evaporated at ambient

temperature, and then rediluted in 100 p.1 of chloroform. If

problems were encountered in the column clean-up step and a

strongly yellow colored solution was observed, the samples

were diluted to 130 p.1 to improve GC separation. The sample

GC injection volume was I pi.

The samples were analyzed using a Hewlett Packard G

I 800A GCD gas chromatograph-electron impact quadrupole

mass spectrometer. A 30-rn long. 0.25 mm internal diameter,

0.25-mm film thickness HP-S MS column was used for the GC

separation. The inlet temperature was 225#{176}C,the detector tern-

perature was 280#{176}C,and the initial column temperature was

I 10#{176}C.The temperature program was: I 10#{176}Cfor 2 mm, then

increase at a rate of I 0#{176}C/mmto I 90#{176}C,hold I 2 mm, increase

at 5#{176}C/mm to 220#{176}C,hold for 2 mm and then increase at

20#{176}C/mmto 260#{176}C.and then hold for 6 mm. Helium was used

as the carrier gas, with a flow rate of I .0 mI/mm. The mass

spectrometer detection parameters for selected ion mode were

in!: 120 and 137 (after 6 mm solvent delay) to 17.0 mm. Ions

in!:: 1 15 and 157 were also monitored between 17.0 and 18.0

mm and exclusively monitored after I 8.0 mm to the end of the

run. The acetylated swainsonine in standards and samples was

identified at approximately 16.3 mm. Identity was confirmed by

ratios of the mn/c I 20: 137 ions and checking the elution profiles

for each ion. Similarly. the primary internal standard, methyl

a-o-mannopyranoside, was detected at I 8.4 mm, using the mmii:

1 15, mmii: 157 ion signals and ratios. The secondary internal

standard, methyl �3-u-galactopyranoside. was detected at I 8.8

mm with ions ma!: I 15 and 157.

Sample and standard concentrations were determined by

integrating the combined signal of the two ions used for detec-

tion. A five point external standard curve. with internal stand-

ards, was included for every 25 samples. Chloroform blanks

were included every 10 samples to ensure that column back-

ground was negligible. The primary internal standard (methyl

a-D-mannOpyranoside) was used as a recovery standard. The

secondary internal standard was used to confirm the primary

internal standard (ratios of signals).

FCM Analysis of PBLs. PBLs were prepared and ana-

lyzed f�r CD3. CD4, CD8. CD14, CDI6, CD2S, CDS7. and

HLA-DR on a Coulter Profile (Coulter). Peripheral blood was

collected in heparin-containing tubes, transported at room tern-

perature, and analyzed within 48 h. Monoclonal antibodies

conjugated with either FIIC or phycoerythrin were purchased

from Becton Dickinson or Coulter: FIIC-labeled t.-PHA was

from E.Y. Labs. Aliquots of 100 p.1 of whole blood were

incubated with anti-CD antibodies or 0. 1 p.g/rnl of FIIC-labeled

L-PHA for 20 mm followed by lysis of RBCs, washing by

centrifugation. and fixation of the cells by using the automated

Q Prep system (Coulter). The lymphocyte population was gated.

and the contaminating monocytes were assessed by staining

with CDI4, which was consistently below 1% of the gated

population.

RESULTS

Twenty-two patients were enrolled in the study. Of these.

19 had a diagnosis of malignancy and 3 were HIV positive.

Three patients (two with malignancy and one with I-IIV corn-

pleted only the pharmacokinetic portion of the study and did not

proceed to biweekly swainsonine treatments. One patient never

received swainsonine treatment. The characteristics of the pa-

tients treated at the four dose levels are summarized in Table I.

Median duration of biweekly swainsonine treatment was 4

weeks and ranged from I to 33 weeks. MID was defined

according to patient toxicities occurring with I month of swain-

sonine treatment as described in Table 2.

MTD Determination. The first three patients enrolled in

each of the dose groups of 50 and 150 p.g/kg twice weekly did

not experience any DLI within the first month of treatment. One

of the three patients in the 300 �gIkg/day dose group experi-

enced DLT after I month (worsening edema). Due to inaccurate

weight measurement, one patient (C. F.) received 417 p.g/kg

biweekly oral doses. This patient was included in the 300 p.g/kg



Table I Clinical characteristics of patients receiving oral biweekly

�- swainsonine

ml

(Total mm = 18)

2.)

2

2

-I

I 0:8

v.eeks.


Disease site

Non-small cell lung

Lung cancerColon cancer

Renal cell cancerBreast cancer

Adrenal cancerMelanomaHypopharyngeal cancer

Hodgkin’s lymphoma

Fibrohistiocytoma

Pancreas cancer

MesotheliomaHIV positive

Males:Females

Age (yr)

Range 25-78

Average 53

Duration of biweekly treatment (weeks) Median

(range)

50 p.g/kg (ii 3) 13 (9-33)

1St) �i.g/kg (mi = 8) 4 (1.5-fl)

3(8) rig/kg (ii = 5)” 10 (1-15)

60)) pg/kg (ml 2)” 4.5 (3-fl)

(, In one of these patients. the dose decreased to I 50 pg/kg after 1

week.

I, In both patients. the dose decreased to 3(8) �gfkg after 2 and 4

dose level for the purposes of MID calculation. Dose escalation

proceeded to 6(X) p.gfkg/day, where the first two patients en-

rolled experienced DLI. One of these patients, N. K. , who had

lung cancer and moderate dyspnea at baseline, developed severe

dyspnea and grade 3 elevation in serum AST after 2 weeks, both

considered possibly related to swainsonine. Patient E. S., with

pancreatic cancer, developed grade 3 elevation in serum AST,

which decreased to grade 2 when the dose was reduced to 300

jig/kg/day for an additional 2 weeks. The drug was then dis-

continued because of obvious disease progression.

As per the requirements of the protocol, three additional

patients were to be enrolled at the next lowest dose to confirm

600 p.g/kg/day as the MID. However, the first two patients

enrolled at the 300 p.glkg/day dose level experienced DLI.

Patient P. S. , with fibrohistiocytoma and numerous nodules in

the lung and moderate dyspnea at baseline, noted an increase in

shortness of breath after the first swainsonine treatment, which

worsened to grade 3 pulmonary edema. Resolution to baseline

status occurred within I week of stopping treatment. During an

episode of disease-related hospitalization for abdominal pain,

patient R. B. was noted to have a grade 3 elevation of serum

ASI. Treatment was interrupted and re-started subsequently at

ISO p.glkg/day for 14 weeks, during which time AST levels did

not exceed grade 2. Thus, in total, two of five patients experi-

enced DLI at the 300 p.g/kg/day dose, and it was, therefore,

considered that the MID had been exceeded at the 600 p�g/kg/

day dose.

Five additional patients were subsequently treated at the

I SO pglkg/day dose. Iwo patients dropped out of the study

because of disease progression prior to completion of 1 month

of treatment. Thus, of the remaining six patients at this dose

level, one experienced DLI. This confirmed 300 p.glkg/day as

the MID. The patient experiencing DLI had lung cancer and

preexisting moderate dyspnea, which worsened to grade 3 dysp-

nea after approximately I month of therapy. Study drug was

discontinued after 6 weeks because of this adverse event.

Treatment-related Adverse Events and Patient Out-

come. An increase in serum ASI during chronic oral swain-

sonine and a decrease in serum AST following cessation of

treatment was observed in all patients as shown for dose level

ISO pg/kg/day in Fig. 2. For all patients, the median AST on

treatment rose to 85 ± 46 units/liter from an average baseline of

45 ± 35 units/liter (P = 0.0001 ). The effect was dose dependent

(P < 0.()01 ). The magnitude of increase in serum AST appeared

to correlate with baseline liver function. Notably, grade 3 AST

abnormalities only occurred in those patients with pretreatment

liver dysfunction. The three patients experiencing grade 3 AST

abnormalities had their swainsonine dose reduced. Two patients

(P. G. and R. F.) exhibited grade 3 elevation in serum bilirubin

based on a single measurement taken after the first 4 weeks of

swainsonine treatment. In both cases, bilirubin returned to nor-

mal within 2 weeks and stayed within the normal range for the

remainder of the treatment period.

Peripheral edema attributed to disease-associated lyrn-

phatic obstruction was present in five patients at baseline. In

three of these patients. edema worsened while on study (see

Table 2). Six patients experienced dyspnea on study (grade 1,

mi = 2; grade 3, ii 3: grade 4, a I ). All of those with dyspnea

more than grade I on study had lungs involved with tumor. As

indicated above, one patient (P. S.) with fibrohistiocytoma met-

astatic to lung exhibited pulmonary edema, probably attributable

to swainsonine, which was reversible when treatment was dis-

continued. In two other patients (A. Z. and I. Y.). the relation-

ship between worsening dyspnea and swainsonine treatment

was unclear. The fourth patient (N. K.) developed grade 4

dyspnea while on study and died within 2 weeks of his last

swainsonine treatment. Autopsy findings ruled out extensive

pulmonary edema and indicated respiratory failure due to dis-

seminated adenocarcinoma, thereby suggesting the observed

shortness of breath was not drug related. Significant, clear

serous exudate was observed weeping from the advanced chest

wall lesions of one subject (V. K.) 1.5 weeks after initiation of

swainsonine treatment.

Two patients (C. F. and R. B.) reported unilateral numb-

ness during swainsonine treatment. In another patient (N. K.),

preexisting lower limb paraesthesia worsened during treatment,

and the patient developed twitching. One of these patients

(C. F.) experienced a 4-day episode of dizziness, tachycardia,

and unsteadiness directly related to cessation of arnitriptyline.

Symptoms resolved within 24 h of amitriptyline reinstitution.

Follow-up brain computed tomography scan investigations were

negative for R. B., but C. F. was found to have a parasagittal

meningioma. Three months following study termination, C. F.

was asymptomatic. R. B. was asymptomatic 2 weeks after the

study.

Other adverse events occurring with a frequency of >20%

(i.e., �4 patients) included fatigue (ti 9), anorexia (ii = 6),

dyspnea (n = 6), and abdominal pain (ii = 4).

At the time of manuscript preparation, six patients were



Dose Ievel/

Patient

Edema Fatigue

Disease site (peripheral) (grade) Dyspnea

ALTAST <35 <4))

units/liter units/liter

Bilirubin <20 Amylase <I 15

mmol/liter mmol/liter Grade”

I: 50 �i.g/kgPG.L.T.R.F.

II: 150 rig/kg

N.H.

E.P.MC.”AZ.V.K.AT.

III: 300 �.Lg/kg

S.O.’,CF.’T.Y.RB.”

P.S.”IV: 600 �J4/kg

N.K.”

ES.”

Lung

Colon

Renal

HIV positiveLung

HIV positiveLung

Breast

Adrenal

Melanoma

Hypopharyngeal

Hodgkin’s

Colon

Fibrohistiocytoma

0 0 0 102 26 27

0 0 0 70 19 14

I 0 0 36 12 19

0 0 0 87 72 20

0 0 0 57 36 13

0 2 0 126 76 14

0 1 3 101 34 16

0 1 0 64 16 13

0 0 I 76 18 16

3 0 0 110 34 19

0 1 0 84 16 17

0 1 2” 76 27 1))0 0 (1 224 85 18

0 1 3 99 67 13

57 1. 0. 2.0

61 1. 0. 0.0

43 1.0.0.0

76 1. 1.0.0

109 1.0.0,0

69 2. 1, 0, 0

109 2. 0. 0, 0

121 1. 0. 0. 1

52 1.0.0.))

l2() 2. 0. 0. I

29 1.0.0.0

152 1.0.0. I

208 3. 1. 0. 2

ND 2. 1. 0. -

Lung 0 1 3 292 63 16 ND 3. 1. 0. -

Pancreatic I’ 0 0 256 86 23 89 3, I. 2. 0

Two patients experienced grade 2 nausea/vomiting. With the exception of M. C. (Kaposi’s sarcoma), HIV patients enrolled in the study may not

have had confirmed cancer. No blood or urine samples were collected from these patients.

“ Pertaining to National Cancer Institute of Canada grading of peak test result indicated in the preceding four colunins as follows: AST.

�2.5 x N = grade I. 2.6-5.0 X N = grade 2. 5.1-20 X N = grade 3: alanine aminotransferase, �2.5 X N = grade I. 2.6-So X N = grade

2, 5.1-20 x N = grade 3: bilirubin, <1.5 >< N = grade 2. 1.5-3.0 X N = grade 3: amylase, <1.5 X N = grade 1. 1.5-2)) X N = grade 2.

2.1-5.0 x N = grade 3.

“ Patients with abnormal liver function tests prior to initiation of swainsonine therapy.

‘ Patients experiencing neurological side effects (numbness in limbs).

“ Shortness of breath present pre-study did not change within the first 4 weeks of swainsonine initiation.

‘. Patients who had dose reduced due to DLT.

I Increased to grade 4 at 6 weeks in therapy.


Table 2 Drug-rekted toxicities occurring within I month ofswainsonine treatment

alive with a median time from final swainsonine treatment of 83

weeks. Twelve patients had died with a median time from last

swainsonine treatment of 14 weeks. Ten of 18 patients were

withdrawn from chronic oral swainsonine treatment because of

disease progression. Eight patients discontinued treatment be-

cause ofadverse events including: fatigue (mm = 3); dyspnea (ii =

3); fatigue, dyspnea. and anorexia (ii = I ): and headache,

numbness, and paraesthesia (ii = I ). Iwo patients discontinued

treatment for other reasons. Disease response was not an end

point of this study. In those patients in whom disease was

evaluable, however, there were no obvious objective responders.

Pharmacokinetics. Sixteen patients were enrolled in this

part of the study at the following doses: SO �i.g/kg (mi = 4): 150

fig/kg (mi = 1 ); 250 �i.g/kg (ii = 5); 500 �i.g/kg (ii = 2): 700

�i.g/kg (ii = 1 ); and 1000 p.g/kg (ii = 2). Plasma level data were

available on 12 of these patients. Thirteen patients entered the

chronic oral study with 5 additional patients enrolled for a total

of I 8 patients. Analysis of swainsonine levels following a single

oral bolus found that 7 of I 2 patients exhibited rapid absorption

of swainsonine with peak plasma levels attained within 3 h of

dosing. Data analysis was limited to C� and T,,1�,, because

swainsonine blood levels did not return to zero within the

sampling period. Table 3 summarizes these two pharmacoki-

netic parameters by dose group and patient. The mean peak

blood levels ranged from 0.3 to 1 .62 jig/ml across the dose

groups. In the upper three dose groups (doses � ISO rig/kg),

C3nax can be considered proportional to dose as indicated by the

approximately constant dose-adjusted C,,,, within the range of

1.3-1.6 (j.i.g/ml)/(�.i.g/kg)J. At the lowest dose (50 p.gfkg). the

dose adjusted maximum concentration was considerably higher.

with a mean value ftr the group of 6. 1 (p.g/mI)/(�g/kg). A

single patient (S. 0.) in the upper dose groups had a higher

dose-adjusted C�, than the three patients in the 50-p.glkg

group. Mean T,�, varied in the range of 160-240 mm with no

obvious dose-relationship.

Inhibition of Golgi and Lysosomal a-Mannosidases.

L-PHA lectin binds to �I-6GIcNAc-branched complex-type

oligosaccharides (35) and. therefore. can be used to monitor

inhibition of the Golgi oligosaccharide processing pathway by

swainsonine (Fig. I ). The glycoproteins synthesized by PBLs

during swainsonine treatment are expected to bear hybrid-type

oligosaccharides rather than the L-PHA-reactive complex-type

structures. In this study. L-PHA binding to PBLs collected

pretreatment and just prior to the month I swainsonine dose

revealed a dose-dependent decrease in � I - #{212}GI cNAc-branched

complex-type oligosaccharides (P = 0.0441: Fig. 3). A transi-

tory increase in L-PHA binding was observed in 5 of 10 patients

at 2 weeks of treatment, notably in patients treated with doses

�300 �gIkg.

Swainsonine inhibits both Golgi a-mannosidase II and

lysosomal a-mannosidases with a similar K of approximately

100 n�i. Blocking the latter enzyme causes accumulation of

oligomannosides in tissues and body fluids (36. 37). Fluoro-

phore-assisted carbohydrate electrophoresis was used to quan-



AST [U/U at 150 �gIkgIday

Thick line: on swainsonine treatment

Thin line: off swainsonine treatment

150

140

130

120

110

100

90

80

70

60

50

40

30

20

10

1082 Phase lB Studs’ of Swainsonine

0 20 40 60 80 100 120 140 160 180 200

TIME FROM START OF SWAINSONINE TREATMENT [DAY]

tify reducing oligosaccharides in urine collected at 2-week in-

tervals froii� five patients as described previously (33). Urine

oligomannosides were detected after 2 weeks on swainsonine

treatment. and levels increased with continued treatii�ent to 4

weeks (data not shown). This suggests that plasma drug levels

attained during the twice weekly dosing schedule are not asso-

ciated with rapid saturation of tissue a-mannosidases as oh-

served previously in the iv. infusion trial, where urine oligom-

annosides reached steady-state concentrations 72 h after starting

swainsonine iiifusion at doses of 150 jig/kg/day or greater (33).

Analysis of PBL Cell Surface Markers. Ten of the I 8

patients who received chronic oral swainsonine had lymphocyte

counts below normal at baseline. which may reflect either un-

derlying advanced malignancy or extensive prior anticancer

treatment characteristic of this study population. In studies in

animals. swainsonine has been shown previously to augment

lymphocyte proliferation (29). No significant changes were

fouiid for hematological parameters in this study. However, a

trend toward an increase in average granulocyte values follow-

ing swainsonine treatment was noted. Mean values increased

33C/( over baseline. Six individual patient profiles showed gran-

ulocyte increases froii� 22 to I 83% over baseline values in the

6-month period (range. 2-21 weeks) following first exposure to

swainsonine.

Swainsonine administered to mice has been shown to in-

crease natural killer cell activity (23 and class II antigen Ia

expression (30. 3 1 ) in heii�atopoietic cell populations. PBLs

collected from eight patients (pretreatment and prior to daily

swainsonine dose on study) were, therefre, examined for

Fig. 2 AST levels during and after cessation of

treatment of eight patients at a dose level of I 5))pgfkg/day swainsonine.

changes in CD3-, CD4-. CD8-, HLA-DR-. CD2S-, CD16-. and

CD 14-positive cells. The total number of CD3 � I-cells express-

ing CD2S (interleukin 2 receptor antigen) was found to decrease

with 2 weeks of oral swainsonine treatment in all patients tested

(Fig. 4). After 1 month of treatment. the percentage of I cells

expressing CD2S had increased from the levels observed at 2

weeks. In two of eight patients. the percentage measured at I

month exceeded pretreati1�ent levels. The ratio of CD4�:CD8�

(i.e.. Ti,,�.iperT kilier/�t,ire��r cell) was demonstrated to increase

with 2 weeks of chronic oral swainsonine treatment in seven of

eight patients tested (Fig. 5). No specific trends were noted in

CD3. HLA-DR. CDI6, or CDI4 expression (data not shown).

DISCUSSION

Upon completion of this second study, adverse events

attributable to swainsonine given by continuous 5-day iv. infu-

sion (as in our previous study; Ref. 33) or chronic twice weekly

oral dosing (described in this study) can be compared as follows.

Serum ASI elevation occurred in both studies but was dose

dependent in the chronic oral schedule only. Pulmonary edema

(with associated dyspnea) and peripheral edema occurred in

both studies, and neurological symptoms were not observed in

the iv. study but did occur in three patients receiving the oral

schedule. From this comparison. it appears that serum ASI

elevation, edema, and dyspnea occur with even short-term ex-

posure to swainsonine, whereas if the neurological side effects

are due to swainsonine, they may be related to repeated dosing

over a prolonged period.



Table 3 Pharmacokinetics: single oral dose of swainsonine

Dose group(�igfkg) Patient

Dosepg/kg

50

5050

C,,,,�”

pg/ml

0.50

0.150.260.30

0.18

C,,,,�fDose

X 10(X)

10.08

3(X)

5.20

6.09

3.62

T,,,,�

(olin)

75

175

300

183

113

50 P. G.

I. T.R. F.Mean

S. D.150-250 1. B.

1. G.A. Z.Mean

S. D.

150

250250

0.25

0.310.280.28

(1.03

1.67

1.241.121.34

0.29

180

50

160163

15

5(X)-7(X) S. 0.

T. Y.C. F.Mean

S. D.

SIX)

5(X)

70))

1.60

0.46

0.4))

0.82

0.68

3.20

0.92

0.57

1.56

1.43

245

>3(M)

175

24()

63

10(X) R. B.N. K.

E. S.

Mean

S. D.

1(X)))1000

1000

l.3()1.8()

1.75

I .62

0.27

I.3()I.8()

1.75

1 .62

0.27

>3(M)I70

2(M)

223

68

Pharmacokinetic data generated from serum drug levels measured at times %llowing a single oral dose of swainsonine as indicated. The serum

drug levels were measured by gas chromatography-mass spectronletry as described in ‘Materials and Methods.” C,,,,�/dose is the dose-adjusted C,,,,�

“ Estimates based On curve fitting.

>3

II)

Ca)

.ECC.a)

40Fig. 3 L-PHA lectin binding to to- �

tal PBLs of patients pre-swainso- �. �

nine treatnleilt (Pre-Tv) and at 2 and �

4 weeks of treatment. L-PHA hind- .E C

ing was measured by FCM as mean �

fluorescence intensity. Results are C �

graphed as percentage of change .� �

from pretreatment levels. Rig/it. pa- 0tients’ initials. �

a)

a)

a

Dose LevelAverage

at 1 Month


Time From Initiation ofTwice Weekly Swainsonine Treatments

Possible mechanisms responsible for swainsonine-induced

toxicities should he considered. Elevated serum AST levels may

be related to swainsonine-induced hepatocyte damage. Increases

in serum GGT (data not reported) observed concomitant with

elevated ASI levels in our iv. study support this explanation.

Furthermore, aminotransferase abnormalities were most severe

in patients with pretreatment liver dysfunction and/or in those

with liver metastases. It has been shown, however, that swain-

sonine is not cytotoxic to cells in tissue culture (15. 16). Alter-

natively, elevated serum ASI levels may result from alterations

in enzyme half-life and clearance caused by swainsonine treat-

ment. Endothelial cells, liver hepatocytes. and macrophages

bind serum glycoproteins via carbohydrate-specific binding re-

ceptors. thereby regulating glycoprotein longevity in the circu-

lation (38). It has been shown that clearance via the mannose

receptor is slower in swainsonine-treated animals (39) and that

slowed clearance of mannose-terminating enzynies is reversible

with discontinuation of swainsonine treatment (40). It is possi-

ble that the increase in circulating mannose-terminating glyco-

proteins due to a-mannosidase II inhibition by swainsonine

(Fig. I ) results in saturation of endocytic and lysosomal path-

ways. causing slower turnover of these enzymes.



U DoseLevelll 15O�.tg/kgA Dose Level Ill 300 �ig/kg

2.5

2

Dose Level IV 600 �.tg/kg T.Y.

(I)

a)

>3

00

0.

E>3

�1+

C.)

aC)+

I’)C,’aC)

C

a)0)C(U

C)

I .5

1

0.5

0Pre-Tx 2 Weeks


I Month

3r

+

a 2.50+

a00li-a)

00=0(U.C

wE.C>3

.Ea)0)C(U

.C0

0�-Pre-Tx 2 Weeks 1 Month


It has been suggested previously that swainsonine may tumor-involved tissues. This mechanism could account for both

cause a capillary leak syndrome (33). Swainsonine has been peripheral and pulmonary edema (causing dyspnea occurring

shown to increase lymphocyte sensitivity to interleukin 2 and with swainsonine and is indicated by the presentation of dysp-

other cytokines (32. 41). A swainsonine-associated capillary nea more than grade I only in those patients with lung involved

leak syndrome. comparable to that which has been documented with tumor. If this hypothesis is correct, we would not expect

in clinical studies using interleukin 2 to treat human malignancy swainsonine to cause either pulmonary edema or significant

(42. 43) could, therefore. be caused by augmented immune cell peripheral edema in patients without underlying malignancy.

reactivity to local cytokines. This effect would be enhanced in Unilateral numbness and paraesthesia were observed only

areas of increased immune cell recruitment and activity such as with chronic oral swainsonine treatment. Neurological symp-


C.F. Fig. 4 CD2S expression on CD3� cells meas-ured as percentage of positive lymphocytes in

peripheral blood at 2 and 4 weeks of treatmentnormalized to pretreatment values. Rig/it. pa-

tieilts’ initials: Pre-Tv. pre-swainsonine treat-

N.K. ment.A.Z.V.K.ES.5.0.A.T.

Fit,’. 5 CD4 ‘ :CD8� ratio of CD3 PBLs follow-

itig 2 and 4 weeks of swainsonine treatment. Re-

suIts have been normalized to pretreatment values.

Right, patients’ initials: Pre-Tv, pre-swainsonine

treatment.

. Dos�lll150�g/kg S�O�A.T.E.S.A.Z.N.K.C.F.V. K.� A Dose Level Ill 300�tg/kg T.Y.

I � Dose Level IV 600 �.tg/kg



linical Cancer Research 1085

(‘ J. J. Lipman. unpublished observations.12. Dennis, J. W., Laferte, S., Waghorne. C., Breitman, M. L., andKerbel, R. S. 31-6 branching ofAsn-hinked ohigosaccharides is directly

toms including lethargy and feed refusal are known to occur in

grazing herds consuming diets rich in natural sources of swain-

sonine and other alkaloids (20, 44). Inhibition of lysosomal

al-3 and al-6 mannosidase by swainsonine is known to result

in accumulation of oligomannosides in tissues and body fluids

(36, 37). In our clinical studies, significant urinary oligoman-

noside levels were detected after iv. and oral administration of

swainsonine. Studies in rodents (45) and primates6 have shown

that oligomannoside storage can occur in the brain in the ab-

sence of neurological symptoms. Further studies are required to

clarify the clinical significance of oligomannoside tissue storage.

The mechanisms responsible for swainsonine-related fa-

tigue observed are not apparent. The lower incidence of fatigue

reported in our iv. trial may be explained by the fact that study

participants were predominantly in bed during treatment. This

ymptom may represent neurological toxicity as discussed above:

however, additional studies are required to determine its cause.

A number of the biological effects of swainsonine observed

in preclinical studies were confirmed in patients receiving

swainsonine treatment. Decreased L-PHA binding reflecting

Golgi a-mannosidase inhibition was documented in both clini-

cal trials. A dose-dependent effect was observed in our oral

study, whereas all doses � 150 �i.gfkg/day maximally inhibited

the enzyme in our iv. trial. Reduced enzyme inhibition observed

with oral administration is likely related to lower maximum

serum levels after twice weekly oral dosing versus continuous

iv. infusion. The mean C1,� following a single 150-250 ig/kg

oral dose measured in the pharmacokinetic portion of this study

was 0.28 p.g/ml, whereas blood levels attained by continuous

iv. infusion with doses of 150-250 �.tgIkg/day were an order of

magnitude higher (4-8 �ig/ml; Ref. 33). The transient increase

in L-PHA binding observed at 2 weeks of oral swainsonine

treatment with doses �300 p.g/kg may indicate that low levels

of swainsonine stimulate lymphocyte activation known to be

accompanied by increased expression of GlcNAc-IV and L-

PHA-reactive �3l-6GlcNAc-branched structures (46). A num-

ber of studies of swainsonine in mice have shown that shorter,

intermittent treatment periods maintain immune stimulation ( I 8.

24, 47). Furthermore, doses producing less than a 20% decrease

in normal N-linked complex type glycosylation are sufficient for

immune stimulation (27). If future studies find that immune

stimulation is the primary mechanism of the clinical efficacy of

swainsonine, then very low doses that only partially inhibit

mannosidases may prove to be optimal for clinical use.

By monitoring lymphocyte populations, we endeavored to

elucidate the immunomodulatory effects of swainsonine admin-

istered according to the continuous iv. and chronic oral sched-

ules. Increased HLA-DR expression was observed in the iv.

study, and decreased interleukin 2 receptor-positive I-cell pop-

ulations and an increased Ti,ci�rI kiiicr/suppressor cell ratios were

observed at 2 weeks in some patients in the oral study. These

findings may result from the initiation of complex cytokine

cascades due to enhanced cellular sensitivity to cytokines in the

presence of swainsonine. Any interpretation of this data must be

cautious. Studies in healthier patients are required to clarify the

immune changes that occur with swainsonine treatment.

In summary, the MID of swainsonine administered ac-

cording to a biweekly oral schedule in patients with advanced

malignancies is 300 gig/kg, based on a grade 3 rise in serum

AST and the occurrence of severe dyspnea progressing to grade

3 pulmonary edema. However, if pulmonary edema were to be

considered a disease-related versus a drug-related phenomena,

then the MID in patients without lung involvement may well be

600 �i.gIkg. Further clinical studies in different patient popula-

tions are required to investigate this possibility. Greater inhibi-

tion is likely to have occurred during peak swainsonine levels

reached soon after drug administration: however, the level of

enzyme inhibition necessary to achieve optimal clinical efficacy

is not clear. In view of these findings as well as the serum ASI

changes and putative cytokine-induced capillary leak syndrome

noted, careful patient selection, alternative dosing schedules,

low starting doses, and close toxicity monitoring will be neces-

sary in planning further clinical trial development of this inter-

esting agent in patients with advanced malignancies.

ACKNOWLEDGMENTS

We would like to thank Cathy Bedlington for secretarial assistance

and Ian Sass for technical assistance.

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