Phase I Clinical Study with 8-Chloro-cAMP and Evaluation ...Vol. 1, 377-384, April 1995 Clinical Cancer Research 377 Phase I Clinical Study with 8-Chloro-cAMP and Evaluation of Immunological

Vol. 1, 377-384, April 1995 Clinical Cancer Research 377

Phase I Clinical Study with 8-Chloro-cAMP and Evaluation of

Immunological Effects in Cancer Patients’

Giampaolo Tortora,2 Fortunato Ciardiello,

Stefano Pepe, Pierosandro Tagliaferri,

Angela Ruggiero,3 Caterina Bianco,

Rosario Guarrasi, Keizaburo Mild,

and A. Raffaele Bianco

Cattedra di Oncologia Medica, FacoltI di Medicina e Chirurgia,Universit#{224} degli Studi di Napoli Federico II, Via Pansini 5, 80131

Napoli, Italy [G. T., F. C., S. P., P. T., A. R., C. B., R. G., A. R. B.],and Terumo Institute of Biomedical Sciences, Nakai, Kanagawa,

Japan [K. M.J

ABSTRACTThe site-selective cyclic AMP analogue 8-chloro-

cAMP (8-Cl-cAMP) is able to inhibit the growth of a wide

variety of cancer cell lines in vitro and in vivo. 8-Cl-cAMP

has been extensively investigated as a new potential anti-

cancer agent and, more recently, preclinical Phase I stud-

ies have been conducted in animal models to study its

toxicity. We have conducted the first Phase I trial with

8-Cl-cAMP to define the maximum tolerated dose, toxic-

ity, plasma drug levels, and immunological effects in pa-

tients with cancers refractory to standard treatments. We

have administered 36 courses of 8-Cl-cAMP to 17 patients

by continous i.v. infusion of the drug for 5 days/week for

2 weeks followed by a 1-week rest period. Six increasing

dose levels, from 0.01 to 0.25 mg/kg/h, were explored.

Drug plasma levels were determined and the expression of

interleukin 2 receptor a, amount of natural killer cells,

and cytolytic activity against K562 cells were measured in

peripheral blood lymphocytes. A grade 4 and a grade 3

increase in serum creatinine and a grade 2 increase in

blood urea nitrogen observed in two patients were the

dose-limiting toxicity. The maximum tolerated dose (0.2

mg/kg/h) determined a grade 1 increase in serum creati-

nine. An increase in calcium levels was observed in sev-

eral patients. The 8-Cl-cAMP plasma concentrations ob-

tamed at the steady state were in the range previously

shown to be effective for cancer cell growth inhibition in

vitro. Interleukin 2 receptor a expression, natural killer

cell number, and cytolytic activity from peripheral blood

lymphocytes were markedly increased after 8-Cl-cAMP

administration at all dose levels. In conclusion, at doses

below the maximum tolerated dose, 8-Cl-cAMP was not

toxic but reached plasma concentrations in the potential

therapeutic range for growth inhibition. Moreover, 8-Cl-

cAMP determined a marked biomodulatory effect and

showed antitumor activity.

INTRODUCTIONThe potential usage of cAMP4 analogues for the therapy of

cancer has been widely discussed in the past two decades (1-3).

However, the lack of selectivity and the high doses required for

the available cAMP analogues have been a major obstacle to the

development of this approach into a sound therapeutic trial. A

renewed interest has been fostered by the recent discovery of a

new class of site-selective cAMP analogues that are able to

modulate the activity of the cAMP-dependent PKA at micro-

molar concentrations (3, 4). The PKA is present in eukaryotic

cells as two different isoforms, PKAI and PKAII, consisting of

a tetrameric structure with two regulatory subunits and two

catalytic subunits. While both PKAI and PKAII share identical

catalytic subunits, they differ in the regulatory subunits which

have been defined RI in PKAI and RI! in PKAII, respectively

(5, 6). It has been shown that PKAI and/or its regulatory subunit

RIa are overexpressed in both cancer cell lines and primary

tumors and are induced by cell transformation with growth

factors such as transforming growth factor a, or oncogenes such

as ras or erbB-2 (7, 8). The increased amount of cAMP binding

proteins, which is mainly due to the overexpression of RIa, has

been recently demonstrated as a marker of poor prognosis in

breast cancer patients (9, 10). Moreover, increased RIa expres-

sion has been found in several nontransformed cells as a phys-

iological response to mitogenic stimuli produced by specific

hormones and/or growth factors (11, 12). On the other hand,

increased RI! or PKAII levels are present in normal differenti-

ated cells and precede the cell growth arrest induced by cAMP

analogues (2, 13).

Unlike intracellular cAMP, the new class of site-selective

cAMP analogues is able to selectively discriminate between the

two cAMP binding sites present on RI and RI! and to modulate

the intracellular levels of these regulatory subunits at micromo-

lar concentrations. 8-Cl-cAMP, the most potent site-selective

cAMP analogue (3, 14), is able to down-regulate RI by inducing

the degradation of the protein while up-regulating at the tran-

scriptional level RIl subunit expression (15). We have shown

Received 9/22/94; accepted 12/27/94.1 This study was supported by the Associazione Italiana per Ia Ricerca

sul Cancro.2 To whom requests for reprints should be addressed.3 Recipient of a fellowship from the Associazione Italiana per Ia Ricerca

sul Cancro.

4 The abbreviations used are: cAMP, cyclic AMP; PKA. protein kinase A;

8-Cl-cAMP, 8-chloro-cAMP; MDR, multidrug resistance; MTh, maxi-

mum tolerated dose; ECOG, Eastern Cooperative Oncology Group; DLT,dose-limiting toxicity; LDH, lactic dehydrogenase; IL-2, interleukin 2;

PBL, peripheral blood lymphocytes; PT!-!, parathyroid hormone.

Research. on March 13, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

378 Phase I Clinical Study with 8-Chloro-cAMP

that down-regulation of RIa by 8-Cl-cAMP or by antisense

oligodeoxynucleotides targeted against the RIa mRNA deter-

mine growth arrest and differentiation in a variety of human

cancer cell lines including, among others, leukemia, breast can-

cer, and colon cancer, with a 8-Cl-cAMP 50% inhibitory con-

centration (IC��) ranging between 0.05 and 5 �LM (3, 7, 8,

15-19). These effects are accompanied by an increased RII:RI

ratio and by inhibition of expression of different oncogenes and

growth factors (7, 8, 16, 20-21). Finally, it has been recently

shown that 8-Cl-cAMP is able to revert MDR in a variety of

MDR cancer cell lines (19).

In vivo studies to evaluate the effect and the pharmacoki-

netics of 8-Cl-cAMP have been performed in mice and dogs and

have shown that the drug reaches the steady-state concentrations

within few hours and that the major route of elimination is rapid

renal excretion; moreover, at doses above 0.34/mg/kg/h on a

30-day-based continuous infusion regimen in dogs, 8-Cl-cAMP

produced gastrointestinal and renal toxicity (22, 23). On the

basis of these studies, to reduce the risk of toxicity and to allow

the recovery of impaired organ function we designed an inter-

mittent exposure regimen of 8-Cl-cAMP administration (see

‘ ‘Patients and Methods’ ‘) and conducted a Phase I clinical trial

in cancer patients refractory to standard therapies.

Previous itt vitro studies suggested that 8-Cl-cAMP might

have a wide spectrum of effects on different cell types. It has

been shown that PKAI/RIa play an important role in the im-

mune response by regulating cytokine production and T lym-

phocyte activation. Moreover, RIa is associated with the T cell

receptor/CD3 complex (24-27). For these reasons we have also

evaluated whether 8-Cl-cAMP administration exerted any effect

on biomodulation in vito, such as immunological changes.

The goals of this trial were: (a) to establish the MTD, (h)

to determine the qualitative and the quantitative toxicities, (c) to

obtain informations on the pharmacokinetics, and (d) to evaluate

the biomodulatory effects on some immunological parameters

of 8-Cl-cAMP administered with this schedule.

PATIENTS AND METHODSEligibility. All patients that entered the study had a his-

tological diagnosis of solid cancers refractory to conventional

therapy. Eligibility criteria included age � 18 years, no cytotoxic

or radiation therapy in the previous 4 weeks, ECOG perfor-

mance status �2, life expectancy of at least 12 weeks, adequate

baseline organ function defined as WBC counts �3,000/pA,

platelets �l00,000/p.l, hemoglobin �9.0 g/dl, total bilirubin

�2.0 mg/dl, aspartate aminotransferase �3-fold normal values,

prothrombine time �60%, creatinine �1.S mg/dl or creatinine

clearance �50 mI/mm, detectable and measurable lesions, and

signed informed consent of the patient.

Drug Dosage and Administration. 8-Cl-cAMP was

synthesized as sodium salt by the Terumo Institute of Biomed-

ical Sciences (Nakai, Kanagawa, Japan). The trial with 8-Cl-

cAMP began at 0.01 mg/kg/h, a dose approximately 30-fold

lower than that producing toxicity in a preclinical study with

dogs (23). 8-Cl-cAMP was administered by continuous iv.

infusion for 5 days a week for 2 weeks followed by 1 week of

rest. The 3-week period was considered one course of therapy.

The daily dose of drug was dissolved in 500 ml dextrose (5%)

in water and infused over 24 h. Toxicity was evaluated accord-

ing to WHO criteria. Unaccetable toxicity was considered grade

3-4 or grade 2 persisting for more than 35 days from the start

of therapy. Additional courses of therapy were administered if

no toxicity was observed during the first cycle of therapy and

with no disease progression. Removal from the study was based

on development of unacceptable toxicity, performance status of

4 (ECOG), refusal to continue the treatment, and disease pro-

gression. The DLT occurring in at least two patients at a given

dose level was used as the end point of the study. The MTD was

therefore defined as the dose level below the DLT.

Patient Monitoring. A complete history, physical exam-

ination, and determination of performance status according to

ECOG criteria was obtained as baseline for each patient. Twelve-

lead electrocardiogram and a chest X-ray were performed at

baseline and at the beginning of each new cycle. Laboratory

studies performed at baseline and three times a week included:

complete blood cell count with leukocyte differential, serum and

urine sodium and potassium, serum calcium, LDH, alkaline

phosphatase, total bilirubin, asparate aminotransferase, alanine

amnotransferase, total protein, and albumin.

Determination of the plasma level concentration of 8-Cl-

cAMP was carried out by HPLC analysis as described below. At

the indicated days of each treatment course, the analysis of the

immunophenotype of lymphocyte subpopulations and the as-

sessment of lymphocyte cytotoxic activity were performed on

10 ml peripheral blood. For immunophenotype studies periph-

eral blood lymphocytes gated using standard FSC/SSC settings

on a FACScan flowcytometer (Becton Dickinson, San Jose, CA)

were analyzed at the indicated time points as described previ-

ously (28). mAbs aleul9 (CD56), uleull (CD16) and IL-2

receptor a (CD2S) (Becton Dickinson, Mountain View, CA)

were conjugated with FITC.

For determination of cytotoxic activity in circulating lym-

phocytes, PBL were isolated from heparinized blood by Ficoll-

Hypaque lymphocyte separation medium (ICN, Costa Mesa,

CA). Fresh PBL were used as effectors in a 4-h LDH release

assay against the human myeloid leukemia K562 cells with a

commercially available cytotoxicity kit (Promega, Madison,

WI; Ref. 29). The assays were performed in triplicate at E:T

ratios between 1 : 1 and SO: 1 . K562 cytotoxicity was expressed as

percentage of specific LDH release calculated according to the

formula:

(LDH release test - effect or spontaneous release)

- spontaneous LDH release

maximum LDH release - spontaneous LDH release

Pharmacokinetic Analysis. Heparinized blood samples

collected at different time points during drug infusion were

centrifuged and plasma was extracted for HPLC quantitative

analysis of 8-Cl-cAMP plasma concentrations. One ml acetoni-

trile was added to plasma and to control plasma + 8-Cl-cAMP,

vortexed, and centrifuged at 3000 rpm for 10 mm. The super-

natant was recovered, allowed to evaporate, reconstituted in

water, filtered, and immediately analyzed by reverse-phase

HPLC, carried out with a Shimazu system. Analytic conditions

consisted of a Tosoh TSK gel column (6 X 150 mm) with an

ammonium acetate (1 M, pH 7.0):H2O:acetonitrile (1:3:6) mo-



Table I Patient characteristics

Patients entered

Male/female

Median age(range)

Performance Status

(ECOG)

17

11/658

(39-73)

0 7

1 8

2 2

CancerLung 2Breast 1Sarcoma 2

Colon 11Mesotelioma 1

Total number of cycles 36

Clinical Cancer Research 379

bile phase pumped at a flow rate of 1 ml/min. Detection was

monitored by fluorescence with emission wavelength set at 260

nm. The retention time of the 8-Cl-cAMP peak was 41.8 mm

and was not present in blank control plasma.

RESULTSSeventeen patients with metastatic malignant tumors re-

ceived 36 courses of treatment. Patients characteristics are de-

scribed in Table 1. The majority (65%) of patients in this trial

had colorectal cancer; all had previously been treated with

cytotoxic therapy for metastatic disease and, at the time of

enrollment, had a performance status (ECOG scale) �2. None

of the patients went off study because of progressive disease or

unacceptable toxicity until the DLT dose level was reached. Six

dose levels were used: 0.01 mg/kg/h (I level), 0.025 mg/kg/h (II

level), 0.05 mg/kg/h (III level), 0.125 mg/kg/h (IV level), 0.2

mg/kg/h (V level), and 0.25 mg/kg/h (VI VI). The number of

patients enrolled for each dose level were two (I level), two (II

level), two (III level), six (IV level), three (V level), and two (VI

level).

Toxicity. The only type of toxicity observed during the

36 courses of treatment was related to alterations of renal

function (Table 2). Most of the patients experienced an increase

in serum creatinine levels at the end of each week of drug

infusion, which, however, remained within normal value range

(Fig. 1A). At the fifth dose level two patients experienced a

grade 1 increase in serum creatinine and one of them a grade 1

proteinuria. At the sixth dose level a grade 4 and a grade 3

increase in serum creatinine and a grade 1 proteinuria occurred

in the two patients studied, while a grade 2 increase in blood

urea nitrogen was observed in one patient (Table 2). The serum

creatinine values for 15 patients are shown in Fig. 1A. In both

patients at the sixth dose level oligoanuria occurred at the end of

the first week of treatment, requiring the discontinuation of drug

infusion. They were both treated with infusion of saline solu-

tions with dopamine at 2.5 �i.gIkg/min, a dose capable of in-

creasing renal perfusion (30). One patient (grade 4) required

extracorporeal dialysis and renal function recovered by the end

of the cycle. The other patient refused the extracorporeal dial-

ysis, thus requiring a longer time for recovery of renal function,

which was reached by day 28. On the basis of these adverse

effects the dose of the sixth level was considered the DLT and

that of the fifth level was the MTh. Interestingly, in all patients

who experienced renal toxicity, no modifications of urine elec-

trolytes were observed, suggesting that the toxic effect may not

be due to impairment of renal perfusion. In one patient enrolled

at the third dose level and in four patients enrolled at the fourth

dose level, repeated cycles of 8-Cl-cAMP (up to six) were

administered. In these cases, several cycles of 8-Cl-cAMP treat-

ment did not influence serum creatinine levels as compared to

the first cycle of treatment, demonstrating that there is no

cumulative renal toxicity (data not shown).

No gastrointestinal, cardiovascular or toxicities other than

renal were observed during the study. Moderate asthenia devel-

oped in patients with an increase in serum creatinine levels.

An increase in total serum calcium, not requiring interrup-

tion of treatment, was observed in several patients after 3-4

days of drug infusion (Fig. 1B). In a few patients it was asso-

ciated with elevation of serum creatinine levels, suggesting a

correlation with the changes in renal function. The measurement

of PTH in two of these patients showed values below the

detectable level (data not shown).

Serial measurements of blood cell counts, hemoglobin con-

centration, and differential WBC counts showed no hematolog-

ical toxicity at any dose level or in patients that received

multiple cycles of treatment (data not shown).

8-Cl-cAMP Plasma Levels. Determination of plasma

concentrations of 8-Cl-cAMP was performed daily in 10 pa-

tients, including at least one patient for each dose level. In

patients enrolled up to the fourth dose level the steady state was

reached rapidly and a moderate increase of drug plasma levels

was present at the end of each week of infusion, returning to

almost basal levels after resting time. In contrast, in the patients

at the fifth and sixth dose levels, in whom renal toxicity oc-

curred in spite of drug infusion discontinuation, the plasma

levels continued to increase also during the resting time, sug-

gesting that impaired renal function might contribute to drug

accumulation. Fig. 2A shows the plasma concentrations of 8-Cl-

cAMP in different patients at day 3 of drug infusion. In Fig. 2B

are shown the pharmacokinetic profiles of two different patients.

The patient enrolled at the fourth dose level did not experience

any toxicity, whereas in the patient receiving the dose of the

fifth level a grade 1 renal toxicity was observed. Since 400 ng

8-Cl-cAMP/ml of plasma correspond to about 1 p.M, it is note-

worthy that in most patients the 8-Cl-cAMP plasma concentra-

tions obtained in the absence of toxicity were within the range

of 8-Cl-cAMP 50% inhibitory concentration (0.05-S p.M) for the

majority of human cancer cell lines tested in vitro (19, 31).

Immunophenotyping and Cytolytic Activity of Circu-lating Lymphocytes. Flow cytometric analysis of the expres-

sion of IL-2 receptor a (CD2S), considered as a marker of

functional activation of T lymphocytes, was performed on the

PBL of eight patients enrolled at the first four dose levels.

Regardless of dose level, all patients analyzed showed a marked

increase in CD2S-expressing cells, peaking between days S and

8 of drug treatment (Fig. 3A). Next we analyzed the effect of

8-Cl-cAMP administration on a natural killer subpopulation of

circulating lymphocytes, as identified by immunoreaction with

anti-CDS6 and anti-CD16 antibodies. As shown in Fig. 3B, we

found a 3- to 4-fold increase of CDS6/16-positive cells at all



A

�0

EVaaVVIi

0

B

�0

0)

E

E

U

00

00

Fig. I Effect of 8-Cl-cAMP treatment on serum creatinine levels (A)

and serum calcium levels (B). Determinations were performed at theindicated time points in the 17 patients evaluated for each cycle of

treatment. Data shown are relative to the first cycle of treatment of 15

patients. Serum calcium values have been corrected on the basis ofalbumin levels. Dotted line, upper limit of normal values.

8 10 12

Days of Treatment

380 Phase I Clinical Study with 8-Chloro-eAMP

Table 2 Toxicit y (WHO) at different dose levels

Dose levels

I Level (4)” II Level (4) III Level (8) IV Level (15) V Level (3) VI Level (2)-

Hematological flh n n

Hemorrhage n n n n n n

Gastrointestinal n n n n n n

Nausea/vomiting n n n n n n

Diarrhea n n n n n n

Renal/bladder

creatinine n n n n Grade I (2/’ Grade 4(1)

Grade 3 (1)Blood urea nitrogen n n n n n Grade 2 (1)Proteinuria n n n n Grade I (1) Grade 1 (2)Fever n n n n n n

Allergic n n n n n n

Other n n n n n n

a Numbers in parentheses, cycles of treatment.

1� n, none.

dose levels, suggesting that 8-Cl-cAMP may contribute to an

expansion of this specific cell population.

To evaluate whether the increased number of CD56/16-

positive cells correlated also with an increased cytotoxic poten-

tial of circulating lymphocytes, we have evaluated the cytolytic

activity using as target the K562 human leukemic cell line.

K562 cytolysis, as determined by specific release of LDH, was

dramatically increased in all patients by day 5 of 8-Cl-cAMP

administration, reaching in some cases values up to 100%

(Fig. 3C).

Antitumor Activity. Although the purpose of this study

was not the evaluation of the therapeutic effect of 8-Cl-cAMP,

we have obtained evidence of antitumor activity in four patients.

In fact, of three patients with advanced colon cancer refractory

to standard chemotherapy with 5-fluorouracil and folinic acid

and that received multiple cycles of 8-Cl-cAMP at the fourth

dose level, one patient exhibited about 70% reduction in a

retroperitoneal lymph nodal mass of 4 x 5 cm after the two

cycles of 8-Cl-cAMP received and progressed 2 months after

completing the treatment. The other two patients had a stable

disease after four cycles of 8-Cl-cAMP with a follow-up of 4

and 7 months, respectively. The remaining three patients of the

fourth dose level, who received up to two cycles of treatment,

showed disease progression 1 or 2 months after the end of

treatment. Furthermore, a patient affected by rapidly progressive

lung adenocarcinoma with several intrathoracic metastatic sites

and that failed to respond to three different regimens of chemo-

therapy was enrolled at the third dose level and no progression

was observed after the administration of six cycles of 8-Cl-

cAMP. Notably, this stabilization of the disease has been lasting

up to 15 months.

DISCUSSION

During the past several years a large amount of experimen-

tal data has been generated on the ability of 8-Cl-cAMP to

selectively modulate the expression of the PKA isoforms, lead-

ing to inhibition of cancer cell growth in vitro and in vivo at

micromolar concentrations (3). Preclinical studies in dogs have

shown that the administration of 8-Cl-cAMP by continuous iv.



APlasma concentntlons of �-a-c�Mi’

Dose L�vthI 11 U m m IV IV IV V VI

PIu.akvth 260 340 400 455 4�I 685 830 700 1200 1790

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0

0.

0

C0U

a0.

H III IV

0 2 4 6 8 10 12 14 16 18 20 22

j

1��

0

0

Time (Days)

Fig. 2 8-Cl-cAMP plasma levels as determined by HPLC analysis. In

A, 8-Cl-cAMP plasma concentations were determined daily. Each num-ber refers to the value obtained at day 3 and represents a different patientat the indicated dose level. Drug infusion was discontinued after 4 days

in patients at the V and VI dose level as renal toxicity appeared. B,

time-concentration profile of 8-Cl-cAMP in two patients enrolled at theIV and V dose levels that are not represented in A. Four hundred ng8-Cl-cAMP/i ml plasma correspond to about I p.M.

infusion for a prolonged time (up to 30 days) allows plasma

levels to reach the micromolar range, with toxic effects mainly

detected in the kidney and the gastrointestinal tract (23). To

reduce the risk of toxicity deriving from a prolonged treatment

we have designed the present Phase I clinical trial introducing

periods of rest between the courses of drug infusion, in order to

allow for host recovery. To determine the MTD and the toxicity

of 8-Cl-cAMP in humans, six dose levels of drug were studied,

starting from a dose approximately 30-fold lower than that

producing toxicity in dogs. Thirty-six cycles of treatment were

administered to 17 patients with cancers refractory to standard

therapies.

The only toxic effect observed consisted of altered renal

function. The DLT was reached at the sixth dose level when a

grade 4 and a grade 3 increase in serum creatinine with protein-

uria occurred in two patients. At the fifth dose level two patients

in one cycle experienced a grade 1 increase in serum creatinine

which returned to basal levels promptly upon drug discontinu-

ation. This was then considered the MTD. Although definitive

data on the mechanism of renal toxicity are not yet available,

toxicology studies conducted in mice and dogs have described

renal tubular necrosis as a major histopathology finding (23). In

our patients the urinalyses have shown proteinuria but not

inversion of the electrolytes, supporting the hypothesis that

8-Cl-cAMP may determine direct toxicity rather than alteration

of renal perfusion. Unlike the study in dogs, which showed

60

40

20

0

Fig. 3 Immunological effects of 8-Cl-cAMP treatment on circulating

lymphocytes. Flow cytometric determination of CD25-positive cells (A)

was carried out on two patients at the I dose level, two patients at the II dose

level, one patient at the III dose level, and three patients at the IV dose level,respectively. Where shown, data represent the average ± SD. Flow cyto-

metric determination of CD56/16-positive cells (B) was carried out on onepatient at the I dose level, one patient at the II dose level, one patient at the

III dose level, three patients at the IV dose level, two patients at the V dose

level, and two patients at the VI dose level, respectively. Where shown, data

represent the average ± SD. Cytolytic activity (C) is referred to one cycle

for each patient. Data represent the average value of two different experi-ments, each performed in triplicate. SD was less than 10%.

gastrointestinal toxicity, no similar effect was observed in pa-

tients of the present study. It has been proposed that RIa/PKAI

are increased in cells that are at an early stage of differentiation


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S A. L. Harris, personal communication.

and/or are rapidly proliferating. Although a recent study by

Pinto et al. (31) on cells from acute myeloid leukemia patients

has shown that 8-Cl-cAMP is able to selectively inhibit the

self-renewal of the leukemic stem cell and therefore may not

directly affect normal hemopoiesis, there has been concern

about the potential hematological toxicity of 8-Cl-cAMP. In this

study we have performed blood cell counts and evaluated he-

moglobin concentration, and no toxicity was observed at any

dose level or following repeated courses of 8-Cl-cAMP.

Several patients experienced an increase in serum calcium

levels at the end of the first week of infusion, which recovered

during the days of rest. Determination of PTH levels in two

patients who did not experience toxicity revealed PTH values

below the detectable levels, suggesting that hormonal regulation

may be implicated for this phenomenon. Similar results were

obtained in a parallel trial at the University of Oxford where an

extensive study of this phenomenon has been conducted.5 It has

been shown that hypercalcemia is often due to PTH-like pep-

tides acting through the cAMP-mediated pathway (32). It is

possible that, at least in some of the patients, 8-Cl-cAMP

determines hypercalcemia with a negative feedback on PTH

secretion by acting downstream to PTH-related molecules.

HPLC-based determination of 8-Cl-cAMP plasma levels

demonstrated that 8-Cl-cAMP administered as continuous infu-

sion rapidly reaches the steady-state concentrations up to the

fourth dose level while, at higher doses, plasma levels increase

after a few days of infusion in parallel to the appearance of renal

toxicity. These results confirm the data previously reported in

mice and dogs and recently described in a study conducted in

humans receiving continuous infusion of 8-Cl-cAMP (33). More

important, our data show that 8-Cl-cAMP at nontoxic doses

reached micromolar concentrations that were in the range of

those effective in producing growth inhibition in the majority of

human cancer cells previously tested (3, 19). Interestingly, un-

like the results obtained in dogs, even at higher dose levels no

gastrointestinal toxicity was observed in our patients. In vitro

studies have shown that, in the presence of fresh bovine serum,

8-Cl-cAMP is cleaved by phosphodiesterases and S’-nucleoti-

dases, resulting in the production of 8-Cl-adenosine (34), a

metabolite which contributes to the growth inhibitory effect of

8-Cl-cAMP (35, 36) but is also responsible for some of the toxic

effects observed in dogs (23). However, it has been recently

demonstrated that human plasma contains a limited amount of

the cleaving enzymes, especially phosphodiesterase III, as com-

pared to cell culture conditions or to other animal models (19),

thus limiting the production of metabolites such as 8-CI-aden-

osine. A recent study conducted by Cummings et al. (33) on

humans treated with 8-Cl-cAMP in continuous infusion con-

firmed the lack of metabolites such as 8-Cl-adenosine in their

plasma. Taken together these results suggest that production of

8-Cl-cAMP metabolites may be different among species, thus

accounting also for differential sensitivity to their toxic effects.

The mechanism of action of 8-Cl-cAMP suggest that it

may have a wide spectrum of effects on different cell types and

compartments. Because of the important role played by PKAI in

the regulation of immune response, we have investigated

whether 8-Cl-cAMP may exert a modulatory effect on the

immune system. We observed that at all dose levels 8-Cl-cAMP

markedly increases the expression of IL-2 receptor a as deter-

mined by measuring the positivity of anti-CD2S-reacting lym-

phocytes. Moreover, measurement of CDS6/1 6-positive cells

showed a marked increase in this specific subset of natural killer

cells, which play a major role in antibody-dependent cell cyto-

toxicity (37). To determine whether 8-Cl-cAMP causes only an

expansion of these cell populations or enhances also their cyto-

lytic potential, we have measured the killing activity of circu-

lating lymphocytes against K562 cells by the use of a LDH

release cytotoxic assay. At all dose levels tested there was an

increase in the cytotoxicity of PBL against K562 cells which, in

some patients, reached values up to 100% LDH-specific release.

Most of the effects observed on the immunological parameters

studied are unrelated to the 8-Cl-cAMP dose, as they occur at all

dose levels. It is likely that 8-Cl-cAMP concentrations obtained

in our patients even at the lower dose levels exceed the mini-

mum required for activation of certain immune effector func-

tions.

We are currently investigating whether these effects are

directly induced by 8-Cl-cAMP on lymphocytic effectors or are

mediated by cytokines released by unidentified cell targets of

the drug. In fact, the modulatory effects of 8-Cl-cAMP strongly

resemble those produced by certain cytokines, such as IL-2 and

interferons (37).

Although the determination of the clinical activity of 8-Cl-

cAMP was not the purpose of our study we have obtained

evidence of antitumor activity in several patients.

Preclinical studies have suggested that 8-Cl-cAMP, an

analogue of a physiological molecule, may be an anticancer

drug through its peculiar action on one of the key mechanisms

of neoplastic transformation (3). In addition, 8-Cl-cAMP is able

to revert the MDR phenotype in several MDR cancer cell lines

(19). From the present study it appears that 8-Cl-cAMP also

produces immunological effects in vivo, which may themselves

turn to be of therapeutic benefit.

To our knowledge the present study is the first Phase I

clinical trial of 8-Cl-cAMP in cancer patients. We have dem-

onstrated that the MTD for the schedule of administration ana-

lyzed is 0.2 mg/kg/h and that toxicity is confined to the kidney.

Pharmacokinetic analysis demonstrated that 8-Cl-cAMP at the

concentration of 0.125 mg/kg/h, a dose devoided of toxicity

corresponding to the fourth dose level, achieves plasma concen-

trations in the potential therapeutic range. Finally, 8-Cl-cAMP

has a clear biomodulatory effect as shown by the changes in

some immunological parameters. The studies conducted thus far

in vitro and in animal models demonstrate that 8-Cl-cAMP is a

cytostatic and a differentiating agent rather than a cytotoxic

drug, suggesting that administration for prolonged time may be

a more successful treatment modality. For these reasons we

believe that a dose completely devoid of any toxicity should be

used for further studies. In this regard, a dose of 0.125 mg/kg/h

may be suggested for a Phase II clinical trial and/or for combi-

nation with other anticancer agents. Such studies are currently in

progress at our institution.




ACKNOWLEDGMENTS

We are indebted to Dr. Y. S. Cho-Chung for her fundamental

findings and leading research on 8-Cl-cAMP that made possible this

clinical work. We thank G. Borriello for excellent technical assistance

and Professor Adrian L. Harris for helpful discussions and critical

reading of the manuscript.

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1995;1:377-384. Clin Cancer Res G Tortora, F Ciardiello, S Pepe, et al. immunological effects in cancer patientsPhase I clinical study with 8-chloro-cAMP and evaluation of

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Phase I Clinical Study with 8-Chloro-cAMP and Evaluation ...Vol. 1, 377-384, April 1995 Clinical Cancer Research 377 Phase I Clinical Study with 8-Chloro-cAMP and Evaluation of Immunological