Phase lb Trial of Bryostatin 1 in Patients with Refractory ...€¦ · may stem from specific patterns of PKC isoform activation (18) or nuclear transbocation ( 19). In addition,

$Page 1: Phase lb Trial of Bryostatin 1 in Patients with Refractory ...€¦ · may stem from specific patterns of PKC isoform activation (18) or nuclear transbocation ( 19). In addition,$
Vol. 4, 611-618, Marc/i 1998 Clinical Cancer Research 611

Phase lb Trial of Bryostatin 1 in Patients with Refractory

Malignancies1

Steven Grant,2 John Roberts, Elizabeth Poplin,

Mary Beth Tombes, Brenda Kyle, Dawn Welch,

Marc Carr, and Harry D. Bear

Departments of Medicine [S. G.. J. R.. E. P.. M. B. T.. B. K.. M. C.],Surgery [H. D. B.l. Biostatistics [D. W.], Microbiology andImmunology [S. 0.. H. D. B.], and Pharmacology [S. G.1, VirginiaCommonwealth University. Richmond, Virginia 23298

ABSTRACT

A Phase lb trial of bryostatin 1, a macrocyclic lactoneand protein kinase C (PKC) activator, was conducted inpatients with refractory nonhematological malignancieswith the primary goal of determining whether down-regu-

lation of peripheral blood mononuclear cell (PBMNC) PKCactivity could be achieved in vivo in humans. Patients (four

patients/cohort) received bryostatin 1 (25 jig/m2) as a 1-h

infusion weekly three times every 4 weeks, but to study the

schedule dependence of pharmacokinetics and pharmacody-

namics, the first dose was administered according to one of

three schedules: (a) a 1-h infusion; (b) a 24-h infusion; or (c)

a split course (12.5 �iWm2 as a 30-mm infusion) on days 1and 4. Conventional toxicities (grades I-Ill) included myal-gias, fever, anemia, fatigue, phlebitis, and headache; in ad-

dition, two patients in cohort 3 experienced transient eleva-

tions in liver function tests, although these patients had

preexisting liver metastases. No objective clinical responses

were encountered. Effects on PBMNC PKC activity wereheterogeneous. Several patients in cohorts 1 and 2 expen-

enced significant declines in activity (-50%) that were sus-tamed in some cases for periods of �72 h. Comparison of

72-h with baseline values for all three patient cohorts corn-

bined revealed a trend toward PKC down-regulation (P =

0.06; signed rank test). For each schedule, plasma bryostatin

1 levels were below the level of detection of a platelet aggre-gation-based bioassay (3-4 nM). Bryostatin 1 administration

failed to produce consistent alterations in lymphocyte im-munophenotypic profiles, interleukin 2-induced prolifera-

Received 5/30/97: revised I 1/25/97: accepted 12/19/97.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadt’ertise,nent in accordance with I 8 U.S.C. Section 1734 solely toindicate this fact.

I Supported by NIH Grants R03 CA66990 and RO1 CA63753 and

Award P30 CA16059 to the Massey Cancer Center and in part by NIHGeneral Clinical Research Center Grant MO1 RR00065. Portions of thiswork were presented in preliminary form at the AACR Annual Meeting,Washington, D.C., April 20-24, 1996.

2 To whom requests for reprints should be addressed, at Division ofHematology/Oncology, Massey Cancer Center, Medical College of Vir-ginia. Virginia Commonwealth University. P. 0. Box 980230, Rich-

mond, VA 23298-0230. Phone: (804) 828-5211: Fax: (804) 828-8079.

tion, or cytotoxicity, although two of three samples from

patients in cohort 3 did show significant posttreatrnent in-

creases in proliferation. Moreover, in some patients, bryo-

statin 1 treatment increased lymphokine-activated killer cell

activity. These findings indicate that bryostatin 1 doses of 25

�ig/rn2 can induce in vivo PBMNC PKC down-regulation inat least a subset of patients and raise the possibility that

higher bryostatin 1 doses may be more effective in achievingthis effect.

INTRODUCTION

Bryostatin 1 is a macrocyclic bactone derived from the

marine bryozoan, Bugula neritina ( 1 ). In addition to immuno-

modulatory actions (2), bryostatin 1 has been shown to exert in

vitro and in vivo activity against a variety of experimental tumor

types, including carcinomas of the breast (3) and lung (4),

melanoma (5), bymphoma (6), and leukemia (7), among others.

In contrast to its inhibitory effects toward neoplastic cells.

bryostatin 1 stimulates the in vitro growth of normal hemato-

poietic progenitors (8), possibly through an indirect mechanism

(9). Based on these and other properties, bryostatin I has been

targeted for clinical development by the National Cancer Insti-

tute, and several Phase I trials have now been completed. When

administered as a weekly 1-h bolus or 24-h infusion, the max-

imum tolerated dose has been reported as 25-50 �ig/m2 (10-

13). Myalgias have been the major dose-limiting toxicity; other

side effects have included fevers, anemia, leukopenia, fatigue,

phlebitis, headaches, dyspnea. hypotension, and bradycardia

(10-13). Limited responses have been observed in patients with

ovarian carcinoma and lymphoma ( 12). Bryostatin I also exerts

in vivo immunomodulatory actions, enhancing expression of

lymphocyte IL-23 receptors and selectively stimulating CD8

cells (13).

Although the mechanism by which bryostatin 1 exerts its

antineoplastic actions remains unknown, considerable evidence

suggests the involvement of the Ca2�- and lipid-dependent

serine-threonine kinase PKC. In in vitro studies using nanomo-

lar concentrations, bryostatin 1 has been shown to bind to and

activate PKC (14). This results in translocation ofthe enzyme to

the membrane, accompanied by depletion of cytosolic activity

(15). In this regard, bryostatin 1 mimics, to a certain extent, the

actions of tumor-promoting phorboids such as phorbol 12-my-

ristate 13-acetate, although its spectrum of activity is distinctly

different from such compounds. For example, bryostatin 1

blocks those phorboid-associated actions that it does not pos-

sess, including tumor promotion in mouse skin ( 1 6) and induc-

tion of differentiation in some leukemic cell sublines ( 15, 17).

3 The abbreviations used are: IL, interleukin: PKC, protein kinase C:PBMNC, peripheral blood mononuclear cell; ara-C, l-�3-o-arabino-furanosylcytosine: LAK, lymphokine-activated killer cell: Ab, antibody.

Research. on December 10, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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612 Phase lb Trial of Bryostatin 1

4 5. Grant and H. D. Bear, unpublished observations.

The basis for the unique actions of bryostatin 1 is obscure but

may stem from specific patterns of PKC isoform activation (18)

or nuclear transbocation ( 19). In addition, prolonged exposure of

cells to bryostatin 1 (e.g., for 24 h) beads to profound reduction

in total cellular PKC activity (20), possibly as a consequence of

proteosomal degradation after ubiquitination (21). This phe-

nomenon, referred to as down-regulation, is distinct from the

early loss of cytosolic activity accompanying PKC activation,

which results from enzyme transbocation to the cell membrane

(22).

Bryostatin 1 has been shown to modulate the apoptotic

response of human leukemia cells to the antimetabolite ara-C

(23). For example, pretreatment of human promyebocytic beu-

kemia cells (HL-60) with bryostatin 1 substantially increases

their susceptibility to ara-C-induced apoptosis, an event tempo-

rally associated with PKC down-regulation (24). This finding is

consistent with reports that PKC activation opposes apoptosis

(25, 26), whereas PKC inhibition promotes cell death (27, 28).

In light of evidence that various chemotherapeutic drugs, in-

cluding ara-C, induce leukemic cell apoptosis in vitro and in

vivo (29, 30), it seems plausible that prior administration of

bryostatin 1 might increase the efficacy of certain antileukemic

agents.

To be effective, this strategy would require that bryostatin

I exert its in vitro actions, i.e. , PKC down-regulation, in vivo.

Currently, little is known about the in vivo effects of bryostatin

I on PKC activity. Recently, we reported that CS7BL/6 mice

receiving a single iv. bolus injection of bryostatin 1 (1 p.g)

experienced significant and sustained PKC down-regulation in

normal splenocytes, an effect accompanied by splenic enlarge-

ment (3 1 ). We have observed that in vitro exposure of normal

PBMNCs to bryostatin 1 results in PKC down-regulation virtu-

ally identical to that displayed by HL-60 cells.4 Thus, PBMNCs

may serve as a surrogate hematopoietic target tissue capable of

mimicking the response of leukemic blasts to this agent. To

assess the potential clinical relevance of these preclinical find-

ings, a Phase lb trial of bryostatin 1 has been conducted in

patients with advanced malignancies. The primary goal of this

trial was to determine whether and to what extent in vivo

administration of bryostatin 1 modulates PKC activity in human

hematopoietic cells.

MATERIALS AND METHODS

Study Design. A fixed dose of bryostatin 1 (25 p.g/m2)

was studied on the basis of experience from previous Phase Ia

trials ( 10-13). Patients received bryostatin 1 (25 pg/m2) as a 1-h

infusion weekly X 3 every 4 weeks; however, the first dose was

administered by random assignment to one of three schedules:

(a) 1-h iv. infusion; (b) 24-h iv. infusion; or (c) 12.5 p.g/m2 as

a 0.5-h iv. infusion on days 1 and 4.

Bryostatin 1 Source and Administration. Bryostatin 1

(NSC 339555) was supplied by the Cancer Treatment and

Evaluation Program, Division of Cancer Treatment, National

Cancer Institute. It was stored at 4#{176}Cin flint vials containing 0.1

mg of bryostatin 1 and S mg of povidone USP lyophilized from

40% t-butanol. Material was reconstituted in 1 ml of sterile PET

(60% polyethylene glycob 400, 30% ethanol, and 10% Tween

80) diluent. The resulting solution was diluted further with 9 ml

of 0.9% sodium chloride. Tubing for the drug infusion was

primed with bryostatin 1 in the PET formulation diluted with

sodium chloride to minimize adsorption of drug. For infusions

of � 1 h, bryostatin 1 was administered by syringe pump via a

peripheral venous line. For 24-h infusions, bryostatin 1 was

administered by syringe pump via either a peripheral venous or

central line with coinfusion of 2 biters of normal saline.

Patient Eligibility. All patients were � 1 8 years of age

and had histologically confirmed lymphoma or solid tumor

malignancy refractory to standard treatment. Other eligibility

criteria included a life expectancy � 12 weeks, Zubrod perform-

ance status � 2, the presence of measurable disease, the absence

of primary or metastatic malignant central nervous system dis-

ease, and no chemotherapy or radiotherapy within 4 weeks of

planned therapy. Laboratory criteria included a WBC in the

range 3.7-15.0 x l034t1, platelets in the range 130-500 X

103/lib, hemoglobin � 10 g/dl, normal protime, bilirubin � 1.5,

normal aspartate aminotransferase and alanine aminotransferase

(�2.5 x the upper limit of normal for metastatic disease), and

creatinine � 1 .5 or creatinine clearance > 70. Pregnant or

nursing patients were excluded from the study.

Toxicity Grading. All patients received an initial evalu-

ation that included a complete physical examination, serum

chemistries, complete blood count, protime, and urinalysis. lox-

icities were graded on a scale of 0-4 according to Cancer and

Acute Leukemia Group B common toxicity criteria, with the

exception of myalgias, which were evaluated based on criteria

previously described by Philip et a!. (10).

Response Criteria. The following criteria were used to

assess the responses of patients to bryostatin 1 : (a) complete

response, absence of all measurable disease, signs, symptoms,

and biochemical changes related to the tumor, including recal-

cification of all lytic bone lesions, for >4 weeks and no new

lesions; (b) partial response, �50% reduction in the sum of the

products of the perpendicular diameters of all measurable le-

sions compared to baseline measurements persisting for �4

weeks, no enlargement of an existing lesion, and no new lesions;

(c) stable disease, neither response nor progression for >8

weeks; and (d) progression of disease, an increase in the product

of the perpendicular diameters of any measured lesion by �25%

compared to baseline measurement or any new lesion.

PBMNC PKC Activity. At designated intervals (prein-

fusion and 1, 8, 24, 48, 72, 96, and 120 h postinfusion), 20 ml

of blood were collected in a syringe containing EDIA (Sigma

Chemical Co., St. Louis, MO) and stored at 4#{176}Cfor not more

than 20 mm before processing. Samples were then diluted 1:2

with sterile RPMI 1640 (Life Technologies, Inc., Grand Island,

NY) and layered over a lO-ml cushion of Ficoll-Hypaque (spe-

cific gravity, 1.077-1.081) in 50-mI sterile polypropylene con-

ical centrifuge tubes. The tubes were centrifuged at 400 X g at

room temperature according to the method of Boyum (32). At

the end of this period, the interface layer containing mononu-

clear cells was extracted with a sterile pipette, transferred to

15-mb plastic centrifuge tubes, and washed twice with fresh

medium.



Clinical Cancer Research 613

Total PKC activity in PBMNC was determined as de-

scribed previously (33) using materials supplied by Life Tech-

nologies, Inc. Briefly, pebleted cells were homogenized in 2 X

10_2 M Iris, S X iO-� M EDIA, and 5 x l0� M EGTA (pH

7.5) containing 25 pg/ml protease inhibitors (aprotinin and

leupeptin), with thirty-five b-s strokes of a motorized mi-

cropestle homogenizer (Glas-Col) at setting 35. The homoge-

nate was incubated on ice for 30 ruin, after which normalized

quantities of protein ( 15 pg) were added to an assay mixture

containing mixed micelbes of phosphatidylserine and phorbol

12-myristate 13-acetate in suspension. The reaction was initi-

ated by addition of 2.5 X b0�� Ci/ml [-y-32P]ATP, 2 X l0� M

nonisotopic AlP, and 5 X b0� M synthetic peptide substrate

(acetylated myelin basic protein amino-terminal peptide

AcMB4_14) in a buffer containing 1 mrvi CaCI, as per the

manufacturer’s instructions. After a 5-ruin incubation at 30#{176}C,

aliquots of the reaction mixture were transferred to phosphocel-

bubose filters, and the reaction was terminated by immersion of

discs in cold 1% (v/v) phosphoric acid. Preliminary studies

established that this reaction was linear over at least a 30-mm

interval. The discs were washed thoroughly, and radioactivity

was quantified by conventional liquid scintilbography. With this

method, replicate determinations generally did not vary by more

than 15%. Baseline pretreatment PKC values for cells obtained

from the 12 patients studied ranged from 1705-4136 prnol

myelin basic protein phosphorybated/mg protein/S mm.

Bryostatin 1 Plasma Assay. Samples were collected in

tubes containing EDTA and centrifuged at 400 X g for 10 ruin

at 4#{176}C,generally within S mm of collection, after which the

plasma was extracted with a sterile Pasteur pipette and trans-

ferred to sterile polypropylene centrifuge tubes. All assays were

performed within 30 mm of blood collection. Bryostatin 1

plasma levels were measured by addition of aliquots of patient

plasma samples to donor whole blood and monitoring AlP

release as described previously (67). Plasma samples (450 p.1)

were mixed with 450 p.1 of whole blood, and after S ruin at room

temperature, 450 p.1 were added to 450 p.1 of 0. 15 M NaCI in an

aggregometer cuvette. Platelet AlP release was measured using

Chrono-Lume reagent and a Chrono-bog whole blood lumi-

aggregometer. Standard curves using known quantities of au-

thentic bryostatin 1 were assayed in parallel and used to deter-

mine concentrations of bryostatin 1 present in plasma samples.

Alternatively, parallel studies were performed assaying the lag

phase of platelet aggregation as described previously (34).

These methods permit detection of plasma bryostatin I levels at

concentrations as bow as S n�i (34).

IL-2 Responsiveness and Lymphocyte Markers. Sam-ples of whole blood were obtained from each patient before

treatment with bryostatin I and at intervals of 24, 72, and 144 h

after initiation of treatment. In cohort 3 (days 1 and 4), day 4

samples were obtained after the second infusion of bryostatin 1.

After density gradient centrifugation as described above, mono-

nuclear cells were harvested, washed, and frozen in FCS + I 0%

DMSO (Sigma). Subsequently, samples were thawed, washed,

and resuspended in complete medium consisting of RPMI 1640,

10% FCS (Hycbone, Logan, UT), 100 units/ml penicillin, 100

p.g/ml streptomycin, 2 msi L-glutamine, 1 m�i sodium pyruvate,

0. 1 msi nonessential amino acids, 10 mtvi HEPES buffer, and

5 x i�-� M �3-mercaptoethanol (Sigma). For proliferation as-

says, cells were plated in triplicate at l0� cells/well in a total

volume of 0.2 ml in 96-well plates (Costar, Cambridge, MA)

with or without 40 lU/mI recombinant IL-2 (Chiron, Emeryville,

CA). After 3 days of culture at 37#{176}Cin a 5% CO,. fully

humidified atmosphere, 0.05 mCi of [3H]thyrnidine (Amer-

sham, Arlington Heights, IL) was added to each well. After an

8-h incubation, cells were harvested onto glass fiber filters using

a PHD Harvester (Cambridge Biotechnology, Inc., Cambridge,

MA), and radioactivity was quantified by liquid scintillography.

Cell samples were also plated at 106 cells/well in 24-

well plates (Costar) under the following conditions: (a) 40

lU/mI IL-2; (b) wells precoated with anti-CD3 monoclonal

Ab (OKT3; Ortho, Raritan, NJ) at 1 p.g/ml, after which the

cells were washed and cultured in the presence of 40 IU/ml

IL-2 as described above; and (c) 500 IU/ml IL-2 alone to

stimulate LAKs. After a 3-day incubation at 37#{176}C.the cells

were harvested, washed, and assayed for cytotoxicity against

target cells using a 4-h 5tCr release assay as described

previously (35). Target cells tested included the P815 murine

mastocytoma, the murine lymphocytic lymphoma EL-4, and

human Daudi cells. Assays involving P815 cells, which have

cell surface Fc receptors, were performed with and without

addition of anti-CD3 monocbonab Ab (1 p.g/ml); in the pres-

ence of anti-CD3, CILs mediate Ab-redirected cytotoxicity

against Fc receptor + targets (36).

For analysis of cell surface markers, PBMNCs were stained

with fluorescein-labeled antibodies directed against CD3, CD4,

CD8, CD2S, CDS6, and DR (MHC class II) and analyzed using

a FACScan analyzer (Becton Dickinson, Sunnyvale, CA) as

described previously (37).

Informed Consent. The study was conducted in accord-

ance with local and federal regulations for the protection of

human research subjects, and all patients offered an informed

consent before enrollment.

Statistical Analysis. PBMNC PKC data for cohorts as a

whole were analyzed by the signed rank test. The significance of

differences between PKC values at individual time points (rel-

ative to baseline control bevels) was determined using Student’s

t test for unpaired observations.

RESULTS

Patient Characteristics. The characteristics of the pa-

tients participating in this trial are summarized in Table 1 . The

I 2 patients included 7 males and 5 females. The median age was

55 years, with a range of 39-76 years. Eastern Cooperative

Oncology Group performance status was 0 in 2 patients and 1 in

10 patients. Disease types and previous therapy were as shown

in Table 1.

Ioxicities. All patients entered on study were available

for evaluation of toxicity. Table 2 lists the mean number of

cycles administered and the associated side effects. In no patient

was treatment discontinued due to bryostatin 1-rebated toxicity.

Patients treated initially according to schedule I (1-h bolus

infusion) experienced myalgias (three of four patients), anemia

(two of four patients). fever (two of four patients), and pruritus

(one of four patients). In all cases, toxicity was grade II or lower.

Myalgias occurred during both early and late treatment cycles

and were responsive to standard analgesic therapy.




Table 1 Patient characteristics

Patient Age (yr) Gender Tumor type Prior treatment

I 49 M Pancreatic cancer Surgery, chemotherapy

2 62 M Pancreatic cancer No prior treatment

3 69 M Melanoma Surgery, chemotherapy4 59 F Non-small cell lung cancer Chemotherapy, radiotherapy

5 62 F Non-small cell lung cancer Chemotherapy, radiotherapy

6 39 F Breast cancer Surgery, chemotherapy, radiotherapy7 54 M Pancreatic cancer Surgery, chemotherapy, hormonal therapy

8 56 F Breast cancer Surgery, chemotherapy, radiotherapy, hormonal therapy, immunotherapy9 66 M Kidney cancer Surgery, chemotherapy

10 44 F Colon cancer Chemotherapy1 1 49 M Melanoma Surgery, chemotherapy, immunotherapy12 43 M Soft tissue sarcoma No prior treatment

Table 2 Toxicities observed

Type of No. ofCohort toxicity Side effect Grade subjects

Cohort 1 HematologicGeneral

AnemiaFeverMyalgia

Pruritus

�2s2�2

I

223

1Cohort 2

Cohort 3

Hematologic

General

GastrointestinalHeartNeurobogic

MetabolicHematologicGeneral

Liver

AnemiaLeukopeniaThrombocytopeniaFeverMyalgiasPhlebitisFullness (head)Performance statusAnorexiaCardiac ectopyMotorHeadacheHypocalcemiaAnemiaFeverMyalgias

Bilirubin

TransaminaseAlkaline phosphatase

�21I

�222I

�3�2

I21122

�2

3

�2�3

41I3111221111113

1

22

Patients initially receiving bryostatin 1 as a 24-h continu-

ous infusion also experienced grade 2 or lower myalgias (one of

four patients), fever (three of four patients), and anemia (four of

four patients). The anemia did not require transfusions and

resolved after discontinuation of treatment. In addition, two of

four patients experienced significant fatigue, leading to a decline

in performance status (to 3) in one patient (grade 3 toxicity).

This symptom resolved after the bryostatin 1 was discontinued

as a consequence of disease progression. Other toxicities en-

countered in this cohort were of grade 2 or lower and included

leukopenia, anorexia, ectopy, thrombocytopenia, phlebitis,

headache, and neuromotor dysfunction.

Patients receiving bryostatin 1 as a 0.5-h infusion (12.5

p.g/m2) on days 1 and 4 (schedule 3) also experienced grade 2

or lower myalgias (three of four patients), fever (one of four

patients), and anemia (one of four patients). In addition, two

patients in this cohort developed grade 3 liver function abnor-

malities, including hyperbilirubinemia (one of four patients) and

elevated alkaline phosphatase bevels (one of four patients).

Grade � II liver function abnormalities included elevated as-

partate aminotransferase (two of four patients), alanine amin-

otransferase (two of four patients), and alkaline phosphatase

(one of four patients). The latter toxicities were self-limited and

resolved following drug discontinuation after disease progres-

sion. It should be noted that each of the patients exhibiting liver

function abnormalities had preexisting liver metastases.

Responses. There were no objective complete or partial

responses in this group of patients. Three patients (with carci-

noma of the pancreas, breast, and soft tissue sarcoma) exhibited

stable disease during treatment that persisted for at beast 8 weeks

before disease progression was noted. Eight patients developed

disease progression during or after the first cycle of therapy, and

one patient elected to discontinue treatment before being eva]-

uated.

PBMNC PKC Activity. The effects of bryostatin 1 on

PBMNC PKC activity are shown in Fig. 1. Patients receiving

bryostatin 1 as a b-h bobus infusion (schedule 1) displayed an

initial increase in PBMNC PKC activity immediately after drug

administration. By 24 h, a decline in PBMNC PKC activity of

50% was observed in two of four patients and -30% in a third

(P � 0.05 in each case). Ihese declines were sustained for

72-120 h, a pattern similar to that previously reported in an

animal model (31). In one patient, PBMNC PKC activity re-

mained elevated at 24 h, fell to pretreatment control levels at

72 h, and subsequently showed a delayed increase in activity

before declining to baseline at 144 h.

Patients receiving bryostatin 1 as a 24-h continuous infu-

sion (schedule 2) showed a heterogenous pattern of PBMNC

PKC activity. Responses at 24 h ranged from a significant

decline in activity (-65%) in one patient, a more modest decline

(-30%) in a second (P � 0.05 in each case), no change in a

third, and a 100% increase in the fourth. The batter patient

subsequently displayed a late decline in activity that approached

50% at 96 h (P � 0.05). Activity returned to control levels at

72-120 h in all but the first patient, whose cells displayed a

more prolonged recovery phase.

Patients receiving bryostatin 1 as a split dose (12.5 p.g/m2

on days 1 and 4; schedule 3) also exhibited a heterogeneous

response. In one patient, a modest decline in PBMNC PKC

activity was observed postinfusion on day 1 (-40%; P � 0.05),

whereas an increase was noted in one of four patients. At 24 h,



Schedule #1(25 p.gIm2; 1-hr infusion)

-.-- Cohort #1

-a-- Cohort #2

-.-- Cohort #3

V.C.9

x

S

C.,

Hours

Schedule #2(25 p.g/m2; 24-hr infusion)

C.)

0.

� t

C.)

0.

�

0.

C.)

0.

0.

Hours post-treatment

-0- Patient #1

-6- Patient #2

-9-- Patient #3

-.-- Patient #4

Patent #1-o’- Pa�ent #2

Patient #3

Patient #4

-a- Patient #1-a-- Patient #2

-9-- Patient #3

-.-- Patient #4

Hours

Schedule #3(12.5p.9/rn2; dl,d4)

0 1 2 3 424 48 72 96 120 144

Hours

activity declined modestly (-30%) in one patient (P � 0.05),

remained unchanged in two patients, and increased (-60%) in

a fourth. After the second dose of bryostatin 1, PBMNC PKC

activity did not change appreciably in three of four patients and

increased in one patient. In this patient, PBMNC PKC activity

remained elevated at the 144-h interval.


Fig. 1 Three cohorts of patients (four patients/cohort) were treatedwith bryostatin 1 (25 �ig/m2) according to three schedules as describedin the text. At the indicated intervals, blood samples were obtained, andmononuclear cell populations were isolated by density centrifugation.After cell disruption, PKC activity was determined by monitoring phos-phorylation of myelin basic protein using a commercially available kit.Values correspond to a percentage of pretreatment controls and areexpressed as the means ± SD for triplicate determinations. Baseline(pretreatment) PKC values for samples obtained from the 12 patientsvaried from 1706 ± 1 15 to 4136 ± 216 pmol phosphorybated myelinbasic protein/mg protein/S mm. *. significantly different from T0 values(P � 0.05).

Fig. 2 Tritiated thymidine uptake by IL-2-stimulated peripheral bloodlymphocytes was monitored in 10 of I 2 patients at the designatedintervals after bryostatin 1 administration as described in the text.Symbols denote the individual patient cohorts. Data are expressed asmean cpm for triplicate wells for each sample after incubation inmedium containing 40 lU/mi IL-2. SDs (data not shown) were < 10% ofthe means in all cases.

For the three patient cohorts as a whole, comparison of 72 h

with baseline PBMNC PKC activities showed a trend toward

down-regulation (P = 0.06; signed rank test). However, no

significant differences among the three cohorts could be iden-

rifled.

Pharmacokinetics Studies. Bryostatin 1 plasma bevels

were undetectable in samples obtained during or after infusion

of bryostatin 1 for all of the schedules used. Based on the level

of sensitivity of the platelet aggregation-based assay (34), this

finding suggests that at the 25 p.g/m2 bryostatin 1 dose level,

plasma bryostatin 1 levels are � S flM.

IL-2 Response and Lymphocyte Markers. Analysis of

phenotypic markers failed to demonstrate consistent changes in

the surface markers of PBMNC (data not shown). For example,

1-25% of PBMNC were CD25+ before treatment, and this

value decreased in 4 of 1 1 patients tested, increased in 2 of 1 1

patients, and remained the same in S of 1 1 patients. In addition,

DR expression, observed in 7-25% of PBMNC before treat-

ment, did not change significantly after administration of bryo-

statin 1 , nor did the expression of other phenotypic markers.

Comparison of phenotypes among different patient cohorts also

did not reveal consistent patterns (data not shown).

When IL-2-stimulated proliferation was assessed, no sig-

nificant change was detected in 7 of the 10 samples assessed at

any time after administration of bryostatin 1 (Fig. 2). However,

in three of the specimens assayed, increases in [3H]thymidine

incorporation ranging from 100-1000% were noted (Fig. 2).

Interestingly, two of the three patients demonstrating an increase

in IL-2-stimulated proliferation were in cohort 3 (split dose),

whereas the third patient was treated with a 1-h infusion of

bryostatin 1.

Lastly, cytotoxicity studies also failed to detect significant

bryostatin 1 effects in the majority of patients (data not shown).

In one patient treated according to schedule 2, Ab-redirected

I-cell cytotoxicity versus P8 1 5 target cells rose from 42%




before treatment to 77% on day 2 (data not shown); values

subsequently declined to basal bevels by day 8. In 3 of 1 1 patient

samples assayed, significant increases in LAK-mediated killing

was observed, most notably on days 4 or 5. An example of such

a response in one of these patients is shown in Fig. 3.

DISCUSSION

Bryostatin 1 has shown a variety of interesting biological

properties both in vitro and in vito. At very low (i.e. , nanomo-

lar) concentrations, it stimulates the in vitro growth of normal

human hematopoietic progenitors (8), possibly through an ac-

cessory cell mechanism (9). Comparable concentrations inhibit

the growth of leukemic progenitors (7, 38), raising the possibil-

ity of a selective antileukemic effect. In animals, bryostatin 1

exhibits activity against a variety of tumor types, including

carcinoma of the breast (3), lung (4). melanoma (5). lymphoma

(6), and in a xenobiotic model, Waldenstrom’s macrogbobuline-

mia (39). It has been assumed that the antineoplastic activity of

bryostatin 1 stems from its ability to activate PKC, although

recent studies bring this concept into question (40). In human

trials to date, bryostatin 1 has been administered by several

schedules, and limited responses have been observed in patients

with malignant melanoma, renal cell carcinoma, and lymphoma

(10-13). The dose-limiting toxicity in each of these studies has

been myalgias, a phenomenon that seems to be cumulative

(10-13). This has led to a recommended Phase II dose of 25-35

p.g/m2 every 1-2 weeks for a 1-h bolus or 24-h continuous

infusion schedule, although recent studies suggest that when

administered as a 72-h infusion, considerably higher bryostatin

1 doses may be achieved (41).

In addition to a potential activity as a single agent, bryo-

statin I may also have a clinical role as a facilitator of neoplastic

cell apoptosis (23, 24). One of the known actions of PKC is to

oppose programmed cell death induced by growth factor depri-

vation (25) or cytotoxic agents (26, 27). Whereas the mecha-

nism by which PKC opposes apoptosis is uncertain, recent

attention has focused on the opposing influences of the PKC and

sphingomyebinase/ceramide pathways on cell viability (42). It

has recently been proposed that factors that modulate the PKC/

ceramide “rheostat” may shift the balance between cell survival

and death (43, 44). The apparent paradox that bryostatin 1 , a

known PKC activator, promotes apoptosis, may be explained by

its delayed ability to induce profound PKC down-regulation

(20) through a mechanism involving ubiquitination and proteo-

somal degradation of the enzyme (2 1 ). Consistent with this

concept, the effect of acute exposure of human leukemia cells to

bryostatin 1 , which leads to increased PKC activity, inhibits

ara-C-induced apoptosis, whereas a more prolonged incubation

(i.e. , 24 h) results in a decrease in enzyme activity and poten-

tiation of apoptosis (24).

In animal studies, in vivo administration of bryostatin 1 has

been shown to induce rapid reductions in cytosolic PKC activity

in peritoneal neutrophibs (45). However, this action, which

stems from transbocation of the enzyme to the cell membrane, is

distinct from the decline in total cellular activity that results

from enzyme down-modulation (22). In a recent study involving

CS7BL/6 mice receiving a single i.v. injection of bryostatin 1 (1

p.g), significant down-regulation of total splenocyte PKC activ-

70

C’,

� 60C.)

I’) 50

�400

�30

�.1 20

10

040:1 20:1 10:1 5:1

Err Ratio

Fig. 3 LAK cytotoxicity against P815 target cells from a single patienttreated according to schedule 2 as described in the text. Data shownrepresent the percentage of specific release from 51Cr-labeled P815target cells by PBMNC effectors after incubation with 500 IU/ml IL-2.Specific release is plotted against E:T ratios. Curves correspond to thenumber of days after bryostatin 1 treatment (shown in the inset). Similar

results were obtained in two additional patients.

ity was observed, which persisted for periods as long as 96 h

(31). This finding indicates that the ability of bryostatin 1 to

induce PKC down-regulation is not restricted to the in vitro

setting. The central goal of this study was to determine whether

in vivo administration of bryostatin 1 in humans by one or more

schedules could reduce PKC activity in a hematopoietic target

tissue, analogous to results obtained in a murine model (3 1).

Once identified, such a bryostatin 1 schedule could then be

combined with a cytotoxic agent (e.g.. ara-C) to mimic in vitro

regimens displaying enhanced antileukemic activity (23, 24).

Although responses in the current trial were quite heterogene-

ous, marked reductions in PBMNC PKC activity were observed

in several patients, and there was a trend (P = 0.06, signed rank

test) toward reduction at 72 h for the three cohorts as a whole.

Both the magnitude and duration of these reductions were

comparable to those encountered in an antecedent murine study

(31). It is possible that down-regulation of PBMNC PKC activ-

ity was limited by the bryostatin 1 dose used in this trial. In this

regard, Jayson et a!. (12) measured total PKC activity in

PBMNC from three patients at various intervals during the

course of a 24-h continuous infusion of 25 p.g/m2 of bryostatin

1. Although changes in PKC activity were noted in these pa-

tients, consistent depletion of activity at the end of the 24-h

infusion could not be documented. Effects on PBMNC PKC

activity at later time points were not examined in that study. It

should be noted that the dose of bryostatin 1 used in this and in

previous trials (25 p.g/m2; Refs. 10-13) has been limited by

cumulative toxicity (chiefly myalgias) that accompanies chronic

administration. It is conceivable that less frequent administra-

tion of bryostatin 1 (e.g., every 4-6 weeks) might permit higher

individual doses to be given, with greater effects on PKC

activity. Studies designed to test this hypothesis are currently

under development.

Plasma bevels of bryostatin 1 , regardless of schedule of




administration, were below the level of detection of the platelet

aggregation-based bioassay, which is sensitive to bryostatin 1

concentrations as low as 2-5 nsi (34). These results, as well as

those of several earlier studies in animals, suggest that the

failure to achieve high plasma bryostatin 1 concentrations stems

from rapid plasma clearance of this compound. For example,

Berkow et a!. (45), using a neutrophib activation assay, reported

that in mice receiving a single i.v. injection of bryostatin 1 (1

�i.g), plasma concentrations declined to undetectable levels (e.g.,

� 60 nM) within minutes of drug administration. More recently,

Zhang et al. (46) described the pharmacokinetics of [C26-

3H]bryostatin 1 (40 p.g/kg) in CD1IF2 mice after iv. or i.p.

administration. When given by the iv. route, btyostatin 1 was

rapidly cleared from the plasma, with concentrations falling to

- 15 nM after 4 h, to < 10 nM after 12 h, and to subnanomobar

concentrations thereafter. Both urinary and gastrointestinal ex-

cretion were detected, and bryostatin 1 was widely distributed in

tissues including lung, liver, heart, lymph nodes, and fat. If the

disposition of bryostatin 1 in humans is similar, rapid plasma

clearance secondary to excretion in conjunction with wide-

spread tissue distribution could account for our failure to detect

plasma bryostatin 1 levels of �5 ntvt in any of the patient

cohorts.

No consistent pattern of alterations in lymphocyte pheno-

type, IL-2-induced proliferation, or cytotoxicity against a van-

ety of targets was observed in the majority of subjects after

treatment with bryostatin 1 . In three patients, however, a striking

increase in IL-2-induced proliferation was noted. Our results

differ somewhat from those of previous trials in which increases

in IL-2 responsiveness and LAK activity were noted (12, 13).

However, in one of these studies, which involved a 24-h infu-

sion schedule, peak increases in IL-2-induced proliferation were

observed 2 h after initiation of drug infusion ( 13). If such

maximal responses typically occur within this time frame, they

would not have been detected at the later intervals examined in

the present study. It may be significant that two of three patients

displaying a significant increase in IL-2-induced lymphocyte

proliferation received bryostatin 1 as a split dose on days 1 and

4. Taken in conjunction with evidence that administration of

bryostatin 1 at or near the maximum tolerated dose in mice

inhibits their resistance to bacterial infection (47), these findings

raise the possibility that lower bryostatin 1 doses may be more

appropriate when this agent is intended as an immunomodulator.

In any case, based on the small number of patients in each

cohort, definitive conclusions regarding the schedule-dependent

immunomodulatory effects of bryostatin 1 cannot be drawn.

In summary, the results of the present study suggest that

administration of bryostatin 1 at a fixed dose of 25 p.g/m2

according to several schedules may be capable of down-regu-

bating PBMNC PKC activity in a subset of patients, and that in

these individuals, down-modulation is qualitatively and quanti-

tatively similar to that observed in a precbinical animal model. In

this study of limited power, response patterns seemed to favor

the 1-h bolus and 24-h infusion over the split-course schedule if

down-regulation of PKC activity is a targeted goal. However,

based on the inconstancy of the PKC modulatory effects, it is

unclear which of the tested schedules will prove optimal for

projected Phase II successor trials. It also remains possible that

higher bryostatin 1 doses could be more effective than the

25-p.g/m2 dose used in the present study. In view of evidence

that bryostatin 1 doses as high as I 20 p.g/m2 are tolerable when

administered as a 72-h continuous infusion (41 ), it is conceiv-

able that such schedules might result in more profound and/or

sustained PKC down-regulation than that observed in the pres-

ent study. Other issues remaining to be addressed include: (a)

whether bryostatin I is capable of down-regulating PKC in

neoplastic target tissues such as leukemic blasts; (b) if so.

whether this action increases the in vivo susceptibility of such

cells to cytotoxic drugs (i.e. , ara-C); and (c) whether the com-

bination of bryostatin 1 and a cytotoxic agent offers the prospect

of improved therapeutic selectivity. To answer these and related

questions, a Phase I trial of escalating doses of bryostatin 1

administered in conjunction with high-dose ara-C in patients

with refractory hematologicab malignancies is currently under

development.

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1998;4:611-618. Clin Cancer Res S Grant, J Roberts, E Poplin, et al. malignancies.Phase Ib trial of bryostatin 1 in patients with refractory

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Phase lb Trial of Bryostatin 1 in Patients with Refractory ...€¦ · may stem from specific patterns of PKC isoform activation (18) or nuclear transbocation ( 19). In addition,