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 Â¡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 cancer. Thus, efforts are being made in many laboratories to by inhibiting protein synthesis, on 54 cancer patients with ad prepare "magic bullets," conjugates consisting of antitumor an vanced disease. Ricin was given as i.v. bolus injections every tibodies and the toxins or their A-chains (12, 17, 22). The two weeks at dose levels ranging from 4.5 to 23 ^g/sq m of possibility exists, however, that, in vivo, the toxic moiety may be estimated body surface area. Ricin was well tolerated at doses released from the conjugate and give rise to general toxic effects up to 18 to 20 u.g/sq m. At these levels and at higher levels, flu- similar to those seen after administration of ricin. It is therefore like symptoms with fatigue and muscular pain appeared and, in important to know the pattern of toxicity of ricin given alone and some patients, nausea and vomiting occurred also. No myelo- the doses of ricin that may produce life-threatening toxicity in suppression was seen. Antibodies to ricin were detected in humans. serum after two to three ricin injections. Ricin was eliminated Because of the demonstrated antitumor activity of ricin in from blood according to first order kinetics. At each dose level, animal models, its unique structure and mechanism of action, its the plasma concentrations, as well as the side effects, showed unusual pattern of side effects, and its potential use in the only minor differences between patients. The highest dose given, preparation of cancerostatic conjugates, a clinical trial seemed 23 /ig/sq m, gave plasma concentrations twice those found warranted. Here we report the results of a Phase I study carried previously to be therapeutically effective in tumor-bearing mice. out at the Norweigan Radium Hospital. Of 38 Ã©valuablepatients, one patient with lymphoma had a partial response. Stable disease was observed in four patients MATERIALS AND METHODS with renal cancers, in two with soft tissue sarcomas, and in one patient each with mesothelioma, thyroid, and rectal cancer. A Patient Selection. During the period from January 1979 to July 1982, dose of 23 ^g/sq m is recommended for Phase II trials of ricin. 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 lympho- INTRODUCTION mas and were no longer candidates for conventional therapy. In 1970, Lin ef al. (13) observed that ricin, a toxic plant protein Forty-one of the patients had solid carcinomas. Of these, 14 were present in castor beans, and the structurally related plant protein renal carcinomas, 12 were gastrointestinal, 4 were head and neck, 2 were lung, 2 were breast, and 2 were testicular carcinomas. One patient abrin, had strong antitumor effects on Ehrlich ascites tumor in had a thyroid carcinoma, 2 had cancers of the esophagus, 1 had cancer mice. Subsequent work in our laboratory demonstrated that ricin of the bladder, and 1 a cancer of unknown origin. Seven of the patients and abrin possess anticancer activity against the murine tumors had sarcomas, 3 had menalomas, 2 had mesotheliomas, and 1 had a Ehrlich ascites, L1210 leukemia, B16 melanoma, and Lewis lung non-Hodgkin's lymphoma. Seven patients received only one course of carcinoma (6, 7), as well as against several human tumor xeno- ricin treatment. The remaining patients had more than 2 courses. grafts in athymic mice (3, 5, 6, 20). In L1210 leukemia, ricin, in Thirty of the patients had been treated previously with both chemo nontoxic doses, was able to potentiate the cancerostatic effect therapy and radiotherapy, 18 had been treated with chemotherapy alone, of doxorubicin, c/s-dichlorodiammineplatinum, and vincristine and 2 had been treated with radiotherapy alone, whereas 4 patients without enhancing the concurrent toxicity (8, 9). Ricin caused were untreated previously. The treated patients had all recovered from only insignificant myelosuppression in mice and in dogs (4, 5). 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 Ricin belongs to a group of structurally related plant proteins mm, and maximum serum creatinine and bilirubin levels were <1.5 mg/ (abrin, modeccin, and viscumin) (18, 19) which have molecular ml. The performance status was >80 on the Karnofsky scale. For all weights of about 64,000 and which consist of 2 polypeptide patients, the expected survival upon entry into the trial was more than 6 chains joined by a disulfide bond. The B-chain is a glycoprotein weeks. which binds the toxin to cell surface receptors, whereas the A- Ricin Preparation: Determination of Plasma Ricin and of Antiricm chain penetrates into the cytosol, where it inactivates the large Antibodies. Ricin was extracted from castor beans (the seeds of Ricinus ribosomal subunit and thus inhibits protein synthesis (16). Communis L, obtained from Deutsche Rizinus-Oelfabrik, Boley and Co., Cellular protein synthesis in vitro is inhibited by ricin in concen Krefelt-Uerdingen, West Germany), as described previously (15). The ricin was purified to homogeneity by chromatography on a CM-52 column trations of approximately 1 ng/ml, and there is evidence that the entry of a single A-chain into the cytosol may possibly be and, subsequently, on a Sepharose-4B column. It was diluted to 10 /Â¿g/ ml in 0.14 M NaCI containing 7 mw sodium phosphate (pH 7.4) and sufficient to kill a cell (1). In vivo, the minimum lethal dose in mice human serum albumin (1 mg/ml; Kabi). was found to be 2.7 ^g/kg and, in dogs, it was found to be 1.75 In the initial studies involving the lower dose levels, we used freeze- (4). dried ricin preparations which were stored in vials and diluted in 0.14 M 1Supported by The Norwegian Cancer Society. NaCI to the desired concentration immediately before use. It was found, 2 To whom requests for reprints should be addressed. however, that the freeze-dried preparations were not stable upon pro Received June 20, 1983; accepted November 2, 1983. longed storage. This difficulty was overcome by freezing solutions of

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Phase l Study of Ricin ricin and storing them in the frozen state at -80Â°. Ricin kept in this state Table 1 was found to be stable, as judged by assays of its toxicity in mice (6). Side effects after ricin treatment After thawing, ricin was used within 2 hr. effectsDose No. of patients Types and severity of side The ricin concentration in blood plasma was determined by a highly sensitive enzyme-linked immunosorbent assay, described in detail else side where.3 The detection limit was approximately 25 pg/ml of serum. Oig/sqm)4.567.5910.512141618202223Total3335531543235WitheffectsNausea00000011 Vomitingpain+â€¢++ Antiricin antibodies and specific IgE were assayed by a similar enzyme- linked 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 +12 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 +2 (1)b++ +4 increments were 1.5 to 2 M9/sq m until more pronounced side effects (1)++ +Muscular (2) ++ were observed, and the increment was then reduced to 1 Â¿ig/sqm. * +, mild; ++, moderate; +++, severe. Three to 5 patients were included at each dose level. At 14 M9/sq m, 6 Numbers in parentheses, number of patients. where we changed from freeze-dried to aqueous preparations, 14 pa tients were entered. >0.99), demonstrate that ricin disappeared from plasma accord Assessment of Toxicity and Tumor Response. Blood cell examina ing to first order kinetics. It was found that the half-lives (tn), tions, carried out twice weekly, included total WBC, differential, RBC, platelet, and reticulocyte counts, as well as measurements of blood urea, calculated from the slopes, were dose dependent and decreased creatinine, uric acid, electrolytes, and serum albumin and liver function with increasing dose up to levels of approximately 20 Â¿tg/sqm, tests. Electrocardiogram and performance status were assessed before where they leveled off. The differences in ricin plasma half-lives the start and at the end of treatment. between individual patients at the same dose level were slight, Temperature, pulse rate, blood pressure, and subjective symptoms with one exception. This patient, who had received 23 M9/sq m were recorded during the 24- to 48-hr hospitalizaron. Tumor response and, concurrently, dexamethasone, had a plasma half-life similar was assessed according to the WHO criteria (23). Only patients with tumors measurable in 2 perpendicular diameters, by palpation, X-rays, to that of the patients who received only 16 M9/sq m of ricin. Antibody Formation. Ricin, being a foreign protein, induces or computed tomography scans, were regarded as Ã©valuable. antibody formation (4, 10). In this study, small amounts of antiricin antibodies could be detected in plasma 3 to 5 weeks RESULTS after the first ricin injection, and the concentration later increased Clinical Side Effects. The side effects, which are summarized rapidly with time. in Table 1, were mild and limited to a few symptom groups and, The immune response to ricin suggested the possibility that at the lower doses, they were almost negligible. Two patients anaphylactic reactions might occur in these patients. However, receiving 14 and 18 M9/sq m of ricin complained of muscular no allergic symptoms were observed, except in the patient who had taken Iscador previously. A blood sample collected before pain. At 20 tig/sq m doses and higher, most patients had fever, and all had flu-like symptoms, with pronounced fatigue and the treatment contained large amounts of antibodies cross- reacting with ricin, and serum collected 10 days after the injection muscular pain. The symptoms usually started 4 to 6 hr after contained small amounts of ricin-specific IgE. In the sera of 22 drug administration and lasted for 1 to 2 days. Some patients other patients tested, no specific IgE was found. had additional symptoms, such as nausea. Four patients at high Antitumor Effect. Among the 38 patients Ã©valuablefor re dose levels vomited for 1 to 2 days. None of the patients got sponse, 1 had a partial response, and 8 patients had stable alopecia, and no phlebitis or extravasation was seen. One of 5 patients at 23 /Â¿9of ricin/sq m who had no side effects had disease. The partial response was seen in a patient with a non-Hodgkin simultaneously received dexamethasone treatment for symp lymphoma. After treatment with 6 ricin doses of 7.5 M9/sq m, a toms caused by brain mÃ©tastases.No significant hematological toxicity was observed, even at the highest dose given. metastasis to the right side of the neck was reduced in volume by more than 50%. The ricin treatment was then discontinued One patient, who had previously taken Iscador, a commercially available extract of mistletoe, developed a widespread skin rash because of high serum antibody titer, and the patient was instead and a swollen tongue and lips immediately after the first dose of given the related protein abrin. She obtained a complete re sponse and has now had a disease-free survival time of 4 years. ricin. These symptoms disappeared gradually, without therapy, in the course of 2 to 3 days. Of the patients with stable disease, 4 had carcinoma of the The biochemical analyses failed to explain the clinical symp kidney which showed no change for a period of at least 6 weeks. Stable disease was also observed in 2 patients with soft tissue toms, as the test results were unaffected by the ricin treatment. Pharmacokinetic Studies. Serum samples were collected sarcoma. Both of them had shown progression during several courses of 4-drug combination treatment. One previously un 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 treated patient with mesothelioma had stable disease for 12 and Methods." In Chart 1, the plasma concentrations at 3 differ weeks during ricin therapy. Subsequently, the disease pro ent dose levels of ricin are plotted against time in a semiloga- gressed rapidly. One patient with rectal cancer, treated previ rithmic plot. The straight lines obtained (correlation coefficients ously with epiadriamycin, showed no growth of liver and lung mÃ©tastasesfor 10 weeks on ricin treatment. One patient with 3 A. Godal, 0. Fodstad, and A. Pihl. Pharmacological studies of ricin Â¡nmiceand papillary thyroid carcinoma had had progression previously dur humans, submitted for publication. ing 8 months. After ricin therapy, the lung mÃ©tastasesshowed

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. 0. Fodstad et al. similar to those reached after minimum lethal dose3 and were twice the levels in mice given therapeutically effective doses (6). r io4 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 IO3 i.v. dose of 23 ng/sq m, administered intermittently every second fi week, will be appropriate in a Phase II trial. C. 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 10 peutic possibilities had been exhausted, only one partial re 12 18 24 sponse was observed. However, indications of effects (stable Time after injection (hr) disease after previous progression) were obtained also in several Chart 1. Elimination of ricin from plasma of cancer patients given different doses other patients. The results suggest that patients with lympho- of ricin. The ricin was given i.v. as a bolus injection. The regression lines were mas, thyroid cancer, soft tissue sarcomas, renal cancers, and determined by the method of least squares (correlation coefficients aO.99). Cone., concentration. 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 no growth during 6 weeks. However, concurrently, a brain me patient had allergic symptoms, and he had previously used the tastasis continued to grow. unregistered drug Iscador, which is known to contain toxic proteins related to ricin (19). The patient had no history of DISCUSSION previous allergic reactions, and the symptoms seemed to be caused by a reaction between ricin and antibodies to Iscador. The present results show that, in the dose range used, ricin With time, antibodies to ricin will counteract and possibly can be administered to cancer patients safely and with moderate abolish the action of the drug. Combinations of ricin and immu- and predictable side effects. These were strictly related to the nosuppressive agents, such as dexamethasone and cyclophos- ricin dose given and, at each dose level, only small differences phamide, delay and reduce the antiricin antibody formation (10). between patients were found, both with respect to the side Hence, combinations of ricin with such compounds may render effects and the pharmacokinetics of ricin. it possible to extend the duration of effective ricin therapy. It is The side effects observed were different from those seen after of interest that there is only slight cross-reactivity between ricin treatment with the drugs most commonly used in cancer treat and the related protein abrin (14). This opens the possibility that ment and, compared with these, they were relatively mild. The abrin may still be effective when ricin has become ineffective due symptoms consisted mainly of fatigue and muscular pain. Since to the presence of high titers of antiricin antibody. This seemed distribution studies have shown that significant amounts of ricin to be the case in one of our patients. cannot be detected in striated muscles (4),3 the myalgias are probably not caused by direct toxic effects on the muscle tissue. ACKNOWLEDGMENTS No bone marrow suppression was detected. There was no alopecia and only insignificant gastrointestinal symptoms. These The technical assistance of Unni Ronning is gratefully acknowledged. findings, together with the observation that ricin selectively po REFERENCES tentiates the antitumor effect of several chemotherapeutic drugs (8, 9), incidate that ricin might be useful in combination chemo 1. Eiklid, K., Olsnes, S., and Pihl, A. Entry of lethal doses of abrin, ricin, and therapy. modeccin into the cytosol of HeLa cells. Exp. Cell Res., 126: 321-326,1980. 2. Fodstad, 0., Aass, N., and Pihl, A. 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Øystein Fodstad, Gunnar Kvalheim, Aslak Godal, et al.

Cancer Res 1984;44:862-865.

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