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[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 CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1984 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1984 American Association for Cancer Research. 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. 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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 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1984 American Association for Cancer Research. Phase I Study of the Plant Protein Ricin Øystein Fodstad, Gunnar Kvalheim, Aslak Godal, et al. Cancer Res 1984;44:862-865. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/44/2/862 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. 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