Download Phase I Study of the Plant Protein Ricin1

[CANCER RESEARCH 44, 862-865, February 1984]
Phase I Study of the Plant Protein Ricin1
Oystein Fodstad,2 Gunnar Kvalheim, Aslak Godal, Jostein Lotsberg, Steinar Aamdal, Herman Host, and
Alexander Pihl
Norwegian Radium Hospital ¡0.F., G. K., J. L, H. H.] and Norsk Hydro's Institute for Cancer Research [0. F., A. G., S. A., A. P.], Montebello, Oslo 3, Norway
ABSTRACT
The high toxicity of ricin and the related 2-chain toxins has
made them candidates for "target-specific" chemotherapy of
A Phase I study was carried out with ricin, a plant toxin acting
by inhibiting protein synthesis, on 54 cancer patients with ad
vanced disease. Ricin was given as i.v. bolus injections every
two weeks at dose levels ranging from 4.5 to 23 ^g/sq m of
estimated body surface area. Ricin was well tolerated at doses
up to 18 to 20 u.g/sq m. At these levels and at higher levels, flulike symptoms with fatigue and muscular pain appeared and, in
some patients, nausea and vomiting occurred also. No myelosuppression was seen. Antibodies to ricin were detected in
serum after two to three ricin injections. Ricin was eliminated
from blood according to first order kinetics. At each dose level,
the plasma concentrations, as well as the side effects, showed
only minor differences between patients. The highest dose given,
23 /ig/sq m, gave plasma concentrations twice those found
previously to be therapeutically effective in tumor-bearing mice.
cancer. Thus, efforts are being made in many laboratories to
prepare "magic bullets," conjugates consisting of antitumor an
Of 38 évaluablepatients, one patient with lymphoma had a
partial response. Stable disease was observed in four patients
with renal cancers, in two with soft tissue sarcomas, and in one
patient each with mesothelioma, thyroid, and rectal cancer. A
dose of 23 ^g/sq m is recommended for Phase II trials of ricin.
INTRODUCTION
In 1970, Lin ef al. (13) observed that ricin, a toxic plant protein
present in castor beans, and the structurally related plant protein
abrin, had strong antitumor effects on Ehrlich ascites tumor in
mice. Subsequent work in our laboratory demonstrated that ricin
and abrin possess anticancer activity against the murine tumors
Ehrlich ascites, L1210 leukemia, B16 melanoma, and Lewis lung
carcinoma (6, 7), as well as against several human tumor xenografts in athymic mice (3, 5, 6, 20). In L1210 leukemia, ricin, in
nontoxic doses, was able to potentiate the cancerostatic effect
of doxorubicin, c/s-dichlorodiammineplatinum,
and vincristine
without enhancing the concurrent toxicity (8, 9). Ricin caused
only insignificant myelosuppression in mice and in dogs (4, 5).
Ricin belongs to a group of structurally related plant proteins
(abrin, modeccin, and viscumin) (18, 19) which have molecular
weights of about 64,000 and which consist of 2 polypeptide
chains joined by a disulfide bond. The B-chain is a glycoprotein
which binds the toxin to cell surface receptors, whereas the Achain penetrates into the cytosol, where it inactivates the large
ribosomal subunit and thus inhibits protein synthesis (16).
Cellular protein synthesis in vitro is inhibited by ricin in concen
trations of approximately 1 ng/ml, and there is evidence that the
entry of a single A-chain into the cytosol may possibly be
sufficient to kill a cell (1). In vivo, the minimum lethal dose in mice
was found to be 2.7 ^g/kg and, in dogs, it was found to be 1.75
(4).
1Supported by The Norwegian Cancer Society.
2 To whom requests for reprints should be addressed.
Received June 20, 1983; accepted November 2, 1983.
862
tibodies and the toxins or their A-chains (12, 17, 22). The
possibility exists, however, that, in vivo, the toxic moiety may be
released from the conjugate and give rise to general toxic effects
similar to those seen after administration of ricin. It is therefore
important to know the pattern of toxicity of ricin given alone and
the doses of ricin that may produce life-threatening toxicity in
humans.
Because of the demonstrated antitumor activity of ricin in
animal models, its unique structure and mechanism of action, its
unusual pattern of side effects, and its potential use in the
preparation of cancerostatic conjugates, a clinical trial seemed
warranted. Here we report the results of a Phase I study carried
out at the Norweigan Radium Hospital.
MATERIALS AND METHODS
Patient Selection. During the period from January 1979 to July 1982,
54 patients, 23 females and 31 males, with a median age of 55 years
(range, 17 to 81) were entered into the study. All patients selected for
the trial had histologically confirmed solid cancers or malignant lymphomas and were no longer candidates for conventional therapy.
Forty-one of the patients had solid carcinomas. Of these, 14 were
renal carcinomas, 12 were gastrointestinal, 4 were head and neck, 2
were lung, 2 were breast, and 2 were testicular carcinomas. One patient
had a thyroid carcinoma, 2 had cancers of the esophagus, 1 had cancer
of the bladder, and 1 a cancer of unknown origin. Seven of the patients
had sarcomas, 3 had menalomas, 2 had mesotheliomas, and 1 had a
non-Hodgkin's lymphoma. Seven patients received only one course of
ricin treatment. The remaining patients had more than 2 courses.
Thirty of the patients had been treated previously with both chemo
therapy and radiotherapy, 18 had been treated with chemotherapy alone,
and 2 had been treated with radiotherapy alone, whereas 4 patients
were untreated previously. The treated patients had all recovered from
major toxic effects of prior therapy, as judged by the laboratory tests.
Their WBC were >4000/cu mm, the platelet counts were 2100,000/cu
mm, and maximum serum creatinine and bilirubin levels were <1.5 mg/
ml. The performance status was >80 on the Karnofsky scale. For all
patients, the expected survival upon entry into the trial was more than 6
weeks.
Ricin Preparation: Determination of Plasma Ricin and of Antiricm
Antibodies. Ricin was extracted from castor beans (the seeds of Ricinus
Communis L, obtained from Deutsche Rizinus-Oelfabrik, Boley and Co.,
Krefelt-Uerdingen, West Germany), as described previously (15). The
ricin was purified to homogeneity by chromatography on a CM-52 column
and, subsequently, on a Sepharose-4B column. It was diluted to 10 /¿g/
ml in 0.14 M NaCI containing 7 mw sodium phosphate (pH 7.4) and
human serum albumin (1 mg/ml; Kabi).
In the initial studies involving the lower dose levels, we used freezedried ricin preparations which were stored in vials and diluted in 0.14 M
NaCI to the desired concentration immediately before use. It was found,
however, that the freeze-dried preparations were not stable upon pro
longed storage. This difficulty was overcome by freezing solutions of
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VOL. 44
Phase l Study of Ricin
ricin and storing them in the frozen state at -80°. Ricin kept in this state
was found to be stable, as judged by assays of its toxicity in mice (6).
After thawing, ricin was used within 2 hr.
The ricin concentration in blood plasma was determined by a highly
sensitive enzyme-linked immunosorbent assay, described in detail else
where.3 The detection limit was approximately 25 pg/ml of serum.
Antiricin antibodies and specific IgE were assayed by a similar enzymelinked immunosorbent assay procedure.
Starting Dose and Dose Escalation. Ricin was administered i.v. every
2 weeks as a bolus injection through the valve in the plastic tubing of an
infusion set while the patient was given 500 ml of normal saline. The
starting dose (4.5 ^g/sq m) was chosen on the basis of toxicity studies
in mice and dogs (4, 5) and was assumed to be equivalent to approxi
mately one-third of the "toxic dose low" (11) in dogs (4). The dose
increments were 1.5 to 2 M9/sq m until more pronounced side effects
were observed, and the increment was then reduced to 1 ¿ig/sqm.
Three to 5 patients were included at each dose level. At 14 M9/sq m,
where we changed from freeze-dried to aqueous preparations, 14 pa
tients were entered.
Assessment of Toxicity and Tumor Response. Blood cell examina
tions, carried out twice weekly, included total WBC, differential, RBC,
platelet, and reticulocyte counts, as well as measurements of blood urea,
creatinine, uric acid, electrolytes, and serum albumin and liver function
tests. Electrocardiogram and performance status were assessed before
the start and at the end of treatment.
Temperature, pulse rate, blood pressure, and subjective symptoms
were recorded during the 24- to 48-hr hospitalizaron. Tumor response
was assessed according to the WHO criteria (23). Only patients with
tumors measurable in 2 perpendicular diameters, by palpation, X-rays,
or computed tomography
scans, were regarded as évaluable.
RESULTS
Clinical Side Effects. The side effects, which are summarized
in Table 1, were mild and limited to a few symptom groups and,
at the lower doses, they were almost negligible. Two patients
receiving 14 and 18 M9/sq m of ricin complained of muscular
pain. At 20 tig/sq m doses and higher, most patients had fever,
and all had flu-like symptoms, with pronounced fatigue and
muscular pain. The symptoms usually started 4 to 6 hr after
drug administration and lasted for 1 to 2 days. Some patients
had additional symptoms, such as nausea. Four patients at high
dose levels vomited for 1 to 2 days. None of the patients got
alopecia, and no phlebitis or extravasation was seen. One of 5
patients at 23 /¿9of ricin/sq m who had no side effects had
simultaneously received dexamethasone treatment for symp
toms caused by brain métastases.No significant hematological
toxicity was observed, even at the highest dose given.
One patient, who had previously taken Iscador, a commercially
available extract of mistletoe, developed a widespread skin rash
and a swollen tongue and lips immediately after the first dose of
ricin. These symptoms disappeared gradually, without therapy,
in the course of 2 to 3 days.
The biochemical analyses failed to explain the clinical symp
toms, as the test results were unaffected by the ricin treatment.
Pharmacokinetic Studies. Serum samples were collected
from 19 patients 3, 6, 12, and 24 hr after injection of 14 to 23
ng of ricin/sq m and assayed for toxin as described in "Materials
and Methods." In Chart 1, the plasma concentrations at 3 differ
ent dose levels of ricin are plotted against time in a semilogarithmic plot. The straight lines obtained (correlation coefficients
3 A. Godal, 0. Fodstad, and A. Pihl. Pharmacological studies of ricin ¡nmice and
humans, submitted for publication.
FEBRUARY
1984
Table 1
Side effects after ricin treatment
effectsDose
No. of patients
Types and severity of side
side
Oig/sq
m)4.567.5910.512141618202223Total3335531543235With
effects
Nausea00000011
Vomiting
pain+•++
+12
++(1)b
+2
+4
+Muscular
++(1)
(2)
++
* +, mild; ++, moderate; +++, severe.
6 Numbers in parentheses, number of patients.
>0.99), demonstrate that ricin disappeared from plasma accord
ing to first order kinetics. It was found that the half-lives (tn),
calculated from the slopes, were dose dependent and decreased
with increasing dose up to levels of approximately 20 ¿tg/sqm,
where they leveled off. The differences in ricin plasma half-lives
between individual patients at the same dose level were slight,
with one exception. This patient, who had received 23 M9/sq m
and, concurrently, dexamethasone, had a plasma half-life similar
to that of the patients who received only 16 M9/sq m of ricin.
Antibody Formation. Ricin, being a foreign protein, induces
antibody formation (4, 10). In this study, small amounts of
antiricin antibodies could be detected in plasma 3 to 5 weeks
after the first ricin injection, and the concentration later increased
rapidly with time.
The immune response to ricin suggested the possibility that
anaphylactic reactions might occur in these patients. However,
no allergic symptoms were observed, except in the patient who
had taken Iscador previously. A blood sample collected before
the treatment contained large amounts of antibodies crossreacting with ricin, and serum collected 10 days after the injection
contained small amounts of ricin-specific IgE. In the sera of 22
other patients tested, no specific IgE was found.
Antitumor Effect. Among the 38 patients évaluablefor re
sponse, 1 had a partial response, and 8 patients had stable
disease.
The partial response was seen in a patient with a non-Hodgkin
lymphoma. After treatment with 6 ricin doses of 7.5 M9/sq m, a
metastasis to the right side of the neck was reduced in volume
by more than 50%. The ricin treatment was then discontinued
because of high serum antibody titer, and the patient was instead
given the related protein abrin. She obtained a complete re
sponse and has now had a disease-free survival time of 4 years.
Of the patients with stable disease, 4 had carcinoma of the
kidney which showed no change for a period of at least 6 weeks.
Stable disease was also observed in 2 patients with soft tissue
sarcoma. Both of them had shown progression during several
courses of 4-drug combination treatment. One previously un
treated patient with mesothelioma had stable disease for 12
weeks during ricin therapy. Subsequently, the disease pro
gressed rapidly. One patient with rectal cancer, treated previ
ously with epiadriamycin, showed no growth of liver and lung
métastasesfor 10 weeks on ricin treatment. One patient with
papillary thyroid carcinoma had had progression previously dur
ing 8 months. After ricin therapy, the lung métastasesshowed
863
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0. Fodstad et al.
similar to those reached after minimum lethal dose3 and were
r
io4
IO3
fi
C.
10
12
Time after
18
24
injection (hr)
Chart 1. Elimination of ricin from plasma of cancer patients given different doses
of ricin. The ricin was given i.v. as a bolus injection. The regression lines were
determined by the method of least squares (correlation coefficients aO.99). Cone.,
concentration.
no growth during 6 weeks. However, concurrently, a brain me
tastasis continued to grow.
DISCUSSION
The present results show that, in the dose range used, ricin
can be administered to cancer patients safely and with moderate
and predictable side effects. These were strictly related to the
ricin dose given and, at each dose level, only small differences
between patients were found, both with respect to the side
effects and the pharmacokinetics of ricin.
The side effects observed were different from those seen after
treatment with the drugs most commonly used in cancer treat
ment and, compared with these, they were relatively mild. The
symptoms consisted mainly of fatigue and muscular pain. Since
distribution studies have shown that significant amounts of ricin
cannot be detected in striated muscles (4),3 the myalgias are
probably not caused by direct toxic effects on the muscle tissue.
No bone marrow suppression was detected. There was no
alopecia and only insignificant gastrointestinal symptoms. These
findings, together with the observation that ricin selectively po
tentiates the antitumor effect of several chemotherapeutic drugs
(8, 9), incidate that ricin might be useful in combination chemo
therapy.
The question arises as to whether the maximum tolerable
dose had been reached in this study. We believe that this is the
case, even though the symptoms observed would ordinarily not
be considered alarming. The myalgias were not intolerable. How
ever, the patients suffered from very pronounced fatigue. Impor
tantly, in an ongoing Phase I study of the related protein abrin,
which has the same mechanism of ation (16) and a similar pattern
of toxicity in animals (4), we have seen 2 toxic deaths. These
patients had general seizures and other symptoms of central
nervous system toxicity at doses which, in other abrin patients,
merely gave symptoms not very different from those here seen
after ricin treatment. This indicates that the dose-survival curve
for abrin and ricin in humans may be very steep, as is the case
in mice (4, 5). Moreover, the plasma concentrations reached in
our patients after the highest dose given (23 nQ/sq m) were
864
twice the levels in mice given therapeutically effective doses (6).
Since mouse data have high predictive value in humans (11),
these findings provide strong evidence that we had reached the
maximum tolerated dose, and we found it inadmissible to raise
the dose further. On the basis of the above data, we feel that an
i.v. dose of 23 ng/sq m, administered intermittently every second
week, will be appropriate in a Phase II trial.
It should be realized that the toxicity and cancerostatic potency
of ricin may differ with the source of the starting material and the
purification procedure used. The activity of ricin preparations
used in humans should therefore be carefully standardized in
biological assays.
In this trial, involving patients in whom all established thera
peutic possibilities had been exhausted, only one partial re
sponse was observed. However, indications of effects (stable
disease after previous progression) were obtained also in several
other patients. The results suggest that patients with lymphomas, thyroid cancer, soft tissue sarcomas, renal cancers, and
rectum cancers should be further studied in Phase II trials.
The antibody formation is a complicating feature in the treat
ment of patients with foreign proteins (21). In our study, only one
patient had allergic symptoms, and he had previously used the
unregistered drug Iscador, which is known to contain toxic
proteins related to ricin (19). The patient had no history of
previous allergic reactions, and the symptoms seemed to be
caused by a reaction between ricin and antibodies to Iscador.
With time, antibodies to ricin will counteract and possibly
abolish the action of the drug. Combinations of ricin and immunosuppressive agents, such as dexamethasone and cyclophosphamide, delay and reduce the antiricin antibody formation (10).
Hence, combinations of ricin with such compounds may render
it possible to extend the duration of effective ricin therapy. It is
of interest that there is only slight cross-reactivity between ricin
and the related protein abrin (14). This opens the possibility that
abrin may still be effective when ricin has become ineffective due
to the presence of high titers of antiricin antibody. This seemed
to be the case in one of our patients.
ACKNOWLEDGMENTS
The technical assistance of Unni Ronning is gratefully acknowledged.
REFERENCES
1. Eiklid, K., Olsnes, S., and Pihl, A. Entry of lethal doses of abrin, ricin, and
modeccin into the cytosol of HeLa cells. Exp. Cell Res., 126: 321-326,1980.
2. Fodstad, 0., Aass, N., and Pihl, A. An inverse relationship between the growth
rate of human melanoma xenografts and their response to some cytostatic
drugs. Br. J. Cancer, 41: 829-831, 1980.
3. Fodstad, 0., Aass, N., and Pihl, A. Response to chemotherapy of human
malignant melanoma xenografts in athymic nude mice. Int. J. Cancer, 25: 453458, 1980.
4. Fodstad, 0., Johannessen, J. V., Schjerven, L, and Pihl, A. Toxicity of abrin
and ricin in mice and dogs. J. Toxicol. Environ. Health, 5: 1073-1084, 1979.
5. Fodstad, 0., Olsnes, S., and Pihl, A. Toxicity, distribution, and elimination of
the cancerostatic lectins abrin and ricin after parenteral injection into mice. Br.
J. Cancer, 34: 418-425,1976.
6. Fodstad, 0., Olsnes, S., and Pihl, A. Inhibitory effect of abrin and ricin on the
growth of transplantable murine tumors and of abrin on human cancers in
nude mice. Cancer Res., 37: 4559-4567,1977.
7. Fodstad, 0., and Pihl, A. Effect of ricin and abrin on survival of L1210 leukemic
mice and on leukemic and normal bone marrow cells. Int. J. Cancer, 22: 558563, 1978.
8. Fodstad, 0., and Pihl, A. Synergistic effect of Adriamycin and ricin on L1210
leukemic cells in mice. Cancer Res., 40: 3735-3739,1980.
9. Fodstad, 0., and Pihl, A. Synergistic effect of ricin in combination with daunorubicin, c/s-dichlorodiammineplatinumfll),
and vincristine in systemic L1210
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1984 American Association for Cancer Research.
VOL.
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Phase I Study of Ricin
leukemia. Cancer Res., 42: 2152-2158,1982.
10. Godal, A., Fodstad, 0., and Pini, A. Antibody formation against the cytotoxic
proteins abrin and ricin in humans and mice. Int. J. Cancer, 32: 515-521,
1983.
11. Goldsmith, M. A., Slavik, M., and Carter, S. K. Quantitative prediction of drug
toxicity in humans from toxicology in small and large animals. Cancer Res.,
35:1354-1364,1975.
12. Jansen, F. K., Blythman, H. E., Carriere, D., Casellas, P., Gros, O., Gros, P.,
Laurent, J. C., Paolucci, F., Pau, B., Pincelet, P., Richer, G., Vidal, H., and
Voisin, G. A. Immunotoxins. Hybrid molecules combining high specificity and
potent cytotoxicity. Immunol. Rev., 62:185-216,1982.
13. Un, J-Y., Tsemg, K-Y., Chen, C-C., Lin, L-T., and Tung, T-C. Abrin and ricin:
new antitumor substances. Nature (Lond.), 227: 292-293,1970.
14. Olsnes, S., Pappenheimer, A. M., Jr., and Meren, R. Lectins from Abrus
precatorius and Ridnus communis. II. Hybrid toxins and their interaction with
chain-specific antibodies. J. Immunol., 113: 842-847,1973.
15. Olsnes, S., and Pihl, A. Different biological properties of the two constituent
peptide chains of ricin, a toxic protein inhibiting protein synthesis. Biochemistry,
12: 3121-3126.1973.
16. Olsnes, S., and Pihl, A. Toxic lectins and related proteins. In: S. van Heyningen
FEBRUARY
1984
17.
18.
19.
20.
21.
22.
23.
and P. Cohen (eds.). The Molecular Actions of Toxins and Viruses, pp. 51105. Amsterdam: Elsevier/North Holland BiomédicalPress, 1982.
Olsnes, S., and Pihl, A. Chimeric Toxins. Pharmacol. Then, 75:355-381,1982.
Olsnes, S., Sandvig, K., Eiklid, K., and Pihl, A. Properties and action mechanism
of the toxic lectin modeccin: interaction with cell lines resistant to modeccin,
abrin, and ricin. J. Supramol. Struct., 9:15-25,1978.
Olsnes, S., Stirpe, F., Sandvig, K., and Pihl, A. Isolation and characterization
of viscumin, a toxic lectin from Viscum album L (misteltoe). J. Biol. Chem.,
257:13262-13270,1982.
Pihl, A., Fodstad, 0., and Olsnes, S. Anticancer properties of the toxic lectins
abrin and ricin. In: Peeters (ed.), Proceedings of XVII Annual Colloquium on
Protides of the Biological Fluids, pp. 631-636. New York: Pergamon Press,
1979.
Sears, H. F., Mattis, J., Heriyn, D., Hägry,P., Atkinson, B., Ernst, C., Steplewski. Z., and Koprowski, H. Phase I clinical trial of monoclonal antibody in
treatment of gastrointestinal tumours. Lancet, 1: 762-765,1982.
Thorpe, P. E., and Ross, W. C. J. The preparation and cytotoxic properties of
antibody-toxin conjugates. Immunol. Rev., 62:185-216,1982.
WHO handbook for reporting results of cancer treatment. Geneva: WHO,
1979.
865
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Phase I Study of the Plant Protein Ricin
Øystein Fodstad, Gunnar Kvalheim, Aslak Godal, et al.
Cancer Res 1984;44:862-865.
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