Altered Tryptophan and Neopterin Metabolism in Cancer ... · tryptophan metabolism and of NEO production after IFN-7 are correlated (8). The effect of IL-2 on IDO activity or on tryptophan

[CANCER RESEARCH 49. 4941-4944. Scpti-mhcr I. I9S9|

Altered Tryptophan and Neopterin Metabolism in Cancer Patients Treated withRecombinant Inter leukin 2'

Raymond R. Brown,2 Carol! M. Lee, Peter C. Kohler, Jaquelyn A. Hank, Barry E. Storer, and Paul M. SondelDepartment of Human Oncology, I'nirersity of Wisconsin Center for Health Sciences, Madison 53792 [R. R. B., P. C. A'., /. A. H., B. E. S., P. M. S.J; Departments ofNutritional Sciences, fC. M. L.] and Statistics ¡B.E. S.J, University of Wisconsin, Madison 53 71)6:and Departments of Pediatrics and (ienetics, ( 'nirenilr of It '¡scansia.

Madison, Wisconsin 53792[P. M. S.f

ABSTRACT

Immune stimulation or Interferon administration induces indoleamine2,3-dioxygenase and GTP cyclohydrolase activity in humans, resulting,respectively, in tryptophan degradation to kynurenine and in neopterinproduction. To determine if similar effects result from interleukin 2 (II2) administration, plasma tryptophan and urinary kynurenine and neopterin nere measured in patients undergoing a phase 1 toxicity trial ofrecomtinant IL-2 given by daily bolus or continuous i.v. administrationfor 7 il ivs at doses of 1 x 10s to I x 10" units/nr/day. Significant dose-

dependent decreases in plasma tryptophan levels and correspondingincreases in urinary kynurenine and neopterin were observed. Thesemetalli lie effects of IL-2 are probably mediated by induction of vinterferon production, although elevated levels of 7-interferon were notfound in the sera of these patients. In view of the indispensable role oftryptor han in synthesis of protein, niacin, and serotonin, we suggest thatsome of the toxic side effects may be the result of this loss of tryptophan.Since these metabolic changes were detected at relatively low doses ofIL-2, these assays provide a highly sensitive means for monitoring invivo metabolic responses to IL-2 therapy.

INTRODUCTION

Infection, lipopolysaccharide administration, or interferontreatment results in the induction of a recently described enzyme, IDO' (EC 1.13.11.17), in a variety of nonhepatic tissues

(1-3). This enzyme is distinct from the liver tryptophan diox-ygenase enzyme in its tissue distribution, molecular weight,immunogenicity, requirement for Superoxide anión,and induc-ibility. Like the liver enzyme, it results in conversion of L-tryptophan to formylkynurenine and subsequent metabolites ofthis pathway.

We recently reported that human recombinant IFN-7 induced tryptophan degradation in vitro in human cell culturesand /> vivo in patients from phase I clinical trials (4-6). UnlikeIFN--), «-and 0-interferon did not induce tryptophan degradation in vitro in human lung or bladder-derived cells (4) buttype I interferons induced IDO in cultures of mixed peripheralblood mononuclear cells (7) and 0-interferon administrationresulted in decreased serum tryptophan and increased urinaryexcretion of KYN (8). These increases in tryptophan catabolismare urdoubtedly the result of the induction of IDO (1-3). IDOis an cxtrahepatic enzyme, distinct from liver tryptophan diox-

ygenase, which opens the indole ring of tryptophan, and to alesser extent of serotonin and certain other Õndoles, usingSuperoxide aniónas a required oxygen source (3, 9). IDO is notinduced by conditions which induce liver tryptophan dioxygen-ase (1-3) and, under conditions which induce IDO, the liver

Received 6/1/88; revised 5/2/89; accepted 5/25/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by NIH Contract BRMP-NOI-CM47669. American Cancer Society Grant CH-237. NIH Grants CA-32685 and RR-03186. and a grantfrom Glaxo Inc.

! To whom requests for reprints should be addressed.*Th<rabbreviations used are: IDO. indoleamine 2.3-dioxygenasc; IFN. inter

feron; IL-2. interleukin 2; KYN. kynurenine: NEO. neopterin: IFN--y, vinter-feron.

enzyme levels are significantly reduced (3). In view of therequirement of IDO for Superoxide anión, it may be significantthat the induction of xanthine oxidase, a potential source ofSuperoxide anión, is another reported effect of IFN-7 (10).

Production and excretion of NEO, a metabolite of GTP, isalso increased in some cancer patients by allogeneic immunestimulation and by IFN or IL-2 administration (8, 11-15). Thisincrease in NEO results from induction of GTP cyclohydrolase(EC 3.5.4.16), which initiates NEO synthesis in macrophages.Although a direct relationship between NEO and tryptophanmetabolism is not known, we have observed that stimulation oftryptophan metabolism and of NEO production after IFN-7are correlated (8).

The effect of IL-2 on IDO activity or on tryptophan metabolism in vivo has not been reported but, since IL-2 stimulatesproduction of IFN-7 in vitro, it might be expected that IL-2would also indirectly induce IDO and GTP cyclohydrolase invivo. The present study was done to assess the effects of IL-2on tryptophan metabolism and NEO excretion in cancer patients receiving IL-2 in a phase I clinical trial and to evaluatethe utility of tryptophan and NEO measurements as indices ofbiologically effective doses of IL-2.

MATERIALS AND METHODS

IL-2 Patients. Patients were those entered into approved protocolsof the University of Wisconsin Clinical Cancer Center for a phase Itrial of IL-2 toxicity. Informed consent was obtained. In accordancewith phase I (toxicity) protocols, a variety of cancer patient types wereeligible for this study (16). Patients in this study included three renalcell, two breast, three lung, three melanoma, and one each of leio-myosarcoma, colon, rectal, pancreatic, parotid gland, synovial sarcoma,poorly differentiated nodular lymphoma, and thyroid tumors. Furtherdetails of this clinical trial have been reported (16). Briefly, patientshad biopsy-proved malignancy which was surgically incurable and forwhich other standard therapies failed or were not known to be effective.Other criteria for eligibility included life expectancy of greater than 4months, age greater than 18 years, blood and urine chemistry withinspecified limits, more than 4 weeks since previous chemotherapy, andno coincident hormonal, immuno-, or radiotherapy.

IL-2 was from Roche Laboratories (Nutley, NJ), as provided by theBiological Response Modifiers Program of the National Cancer Institute, which supported these studies. IL-2 was given i.v. as a bolus over15 min or continuously over 24 h for 7 consecutive days in doses of 1x 10*. 1 x IO6,3 x 10". and 1 x 10" units/irr/day. Three patients were

treated at each dose level and by each route of administration, exceptthat only two patients were treated at the 3 x IO6continuous level and

only two patients received the highest dose of 1 x 10 units/nv (one byeach route), because of undue toxicity requiring that the treatment bediscontinued after 4 days. Complete 24-h urine samples were collectedbefore treatment, daily during the week of treatment, and at 1 and 3weeks after treatment was stopped. Heparinbed blood samples forplasma preparation were taken between 7 and 8 a.m. after overnightfast before treatment, at 2 and 4 h after initiation of treatment, between7 and 8 a.m. daily thereafter during the week of treatment, at 2 and 4h after the last dose on day 7. and at I and 3 weeks after treatment wasstopped. Plasma and urine samples were fro/en promptly and stored at-20°Cuntil analyzed.

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IL-2 EFFECTS ON TRYPTOPHAN AND NEOPTERIN METABOLISM

Analytical Methods. Plasma tryptophan was measured by the fluo-rometrie method of Denkla and Dewey (17) with internal recoverystandards of L-tryptophan. In some cases, this method was confirmedby high pressure liquid chromatography assay of tryptophan (18).Urinary KYN was measured by the alkaline steam distillation method,using either manual or autoanalyzer methods developed in this laboratory (19). Urinary NEO was assayed without further oxidation byminor modifications of the high pressure liquid chromatographymethod of Hausen et al. (20). Routine blood chemistries and patientclinical performance status were monitored.

Statistical Analyses. Changes in tryptophan, KYN, and NEO betweenpretreatment and posttreatment times were compared by paired t testanalyses using arithmetic and logarithmetic methods. Comparisonsbetween pretreatment and posttreatment values were made by t testsusing a log ratio method. Regression analyses of dose versus metabolitelevels were done on data from day 3 (after three doses of IL-2), whichwas the last day before discontinuance of the highest dose and the timewhen most consistent changes occurred in individual patients.

RESULTS

In daily values for the parameters studied here, no significantdifferences were found between bolus and continuous routes ofadministration of IL-2; therefore, data from both groups ofpatients were pooled for further study. Fig. 1 shows the changesin plasma tryptophan in the pooled data from subjects receivingthe four dosage levels. A dose of IO5 units/nr/day did notchange tryptophan levels, but higher doses produced a dose-dependent decrease which had not fully returned to pretreatment levels by day 15. Analyses of samples taken 2 and 4 hafter the first dose of IL-2 on day 0 and after the last dose onday 6 showed that decreases in plasma tryptophan occurredwithin 4 h of IL-2 administration, particularly in patients giventhe 15-min bolus injections. There was a partial restoration oflevels in samples taken just before the next daily bolus injection(data not shown). Excretion of the tryptophan catabolite KYNin 24-h urine samples showed dose-related increases corresponding to the decreases in plasma tryptophan (Fig. 2). Aftercessation of IL-2 administration, KYN levels returned to nearpretreatment values with the exception of the group receivingthe maximum tolerated dose of 3 x IO6units/m:/day, in which

case levels were still significantly elevated on day 15. NEO, aGTP metabolite elevated in a variety of patients having stimulation of the immune system (11-14), was significantly elevatedin urine samples following IL-2 administration (Fig. 3).

Dose-response relationships were assessed using data from

80

34567DAY OF STUDY

15

Fig. I. Average plasma tryptophan levels (^mol/liter) in grouped subjectsgiven bolus or continuous injections of IL-2 for 7 days, except that the subjectsgiven IO7units received only four doses. Doses were based on units of IL-2/m2/day and were 10' (Ü),10' (A), 3 x 10" (•),or IO7(O). Bars, SD.

<

>- DCoc a<u.1°5s?

1200

1000-

800-

600-

400-

200-

-O- DOSE105-•-DOSE106

-•-DOSE 3X106

0 1 0DAY OF STUDY

Fig. 2. Average urinary KYN changes, shown as percentage of pretreatmentvalues. * Time points significantly differing from pretreatment values (P < 0.05).

DOSE 105OOSE106

DOSE 3X106DOSE107

1 0 1 5DAY

Fig. 3. Urinary excretion of neopterin following administration of IL-2 bybolus and continuous routes. Values are expressed as ^mol/24 h. * Valuessignificantly different (P < 0.05) from pretreatment levels.

10° 3X10" 10'

DOSE OF IL-2/DAY (UNITS/M2)

Fig. 4. Relationships of plasma tryptophan (D), urine KYN (•),and urinaryNEO (O) with dose of IL-2 after three doses. Bars, SD.

day 3, since this was the last day that patients receiving 10 xIO6 units of IL-2 were able to tolerate a full dose. These data

are shown in Fig. 4. Significant dose-related changes wereobserved for plasma tryptophan (P = 0.0001), for urinary KYN(P = 0.0001), and for urinary NEO (P = 0.005). The linesshown in Fig. 4 suggest that there is a sharp response betweendoses of IO5and IO6units/nr/day but that higher doses elicited

little further change. However, because of interpatient variability and the limited number of subjects available in this study,this plateau of response could not be established statistically.

In view of the evidence that IFN-7 induces both IDO activityand NEO synthesis in various in vitro systems and that IL-2can induce IFN-7 production, it seems likely that the metabolicchanges observed here were due to the stimulation of IFN-7production by exogenous IL-2. Consequently, plasma IFN-7levels were measured in two independent laboratories but noneof the samples tested had IFN-7 levels consistently abovebaseline; however, it remains possible that IFN-7 was producedin vivo at levels with biological activity in the tissues where itwas produced but was not detectable in the plasma by the

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methods used. Additional evidence that IFN-7 is induced is theobservation that urinary NEO levels were also markedly elevated {Figs. 3 and 4). Synthesis of NEO by macrophages isbelieved to be induced specifically by IFN-7 (10).

These dose-dependent changes in Trp, KYN, and NEO levelscorrespond to dose-dependent alterations in numerous clinicaland immunological parameters observed in these patients andreported elsewhere (21-26). IL-2 could be well tolerated for 4-7 dayi. at doses of 1-3 x IO6 units/nr by either bolus or

continuous infusion (21,22). A striking rebound lymphocytosis,consisting of lymphocytes bearing activation antigens (la, Leu17), was observed within 24 h after stopping of IL-2, with agreater rebound observed after continuous infusions than afterbolus injections (22). Lymphocytes from this time of lympho-cytosis have augmented IL-2 proliferative responsiveness, natural killer cell activity (16), and lymphokine-activated killer cellactivity (23-25). Prolonged treatment for 4 weeks with IL-2,using a similar dose and schedule, induced reproducible, measurable antitumor responses in 3 of 23 treated patients (26).

DISCUSSION

The in vivo induction of tryptophan metabolism in humansby IL-'.l treatment reported here supports and extends the initial

reports (1-3) of induction of IDO activity in mouse tissues byIFN (type 1) or by virus infection, bacterial lipopolysaccharide,or tumor growth in an allogeneic host. Stimulation of NEOexcretion in our subjects is also consistent with reports thatimmune-stimulated patients excrete elevated NEO and that itsmeasurement may be a useful early marker of transplant rejection and other immune-related events (10-15). In the presentstudy with IL-2, changes in urinary KYN are as large or largerthan tliose of NEO and suggest that KYN excretion may alsobe an early, sensitive, and reliable marker of immune stimulation. The decreased plasma tryptophan observed in the presentstudy :s most likely the result of enhanced tryptophan catabo-lism via IDO activity, rather than the result of a poor tryptophanintake In the latter case (27), we observed decreases in urinaryKYN rather than increases as found in the present studies.

Earlier studies from this laboratory and others reported disturbances of tryptophan metabolism in a variety of cancerpatients (28-30) and in patients with certain autoimmune diseases such as rheumatoid arthritis, scleroderma, and Wegner's

granulomatosis (31-33). Prior to the discovery of the IDOenzyme, it was believed that certain of these abnormalities oftryptophan metabolism were the result of stress or hormonalinduction of liver tryptophan degradation or due to a functionaldeficiency of vitamin B-6 causing a block in further KYNmetabolism (34). With the discovery of IDO and its inductionby IFN, and with reports of elevated IFN levels in severalautoimmune diseases (35), it now seems most likely that manyof the previously reported abnormalities of tryptophan metabolism were the result of IFN-induced IDO activity rather thanenhanced activity of the liver enzyme (36).

In view of the essential nature of tryptophan, not only forprotein synthesis but also for synthesis of the neuroimmuno-

modulator serotonin and the vitamin niacin, it is important toconsider the possible physiological and nutritional implicationsof the marked decrease in plasma tryptophan observed in thepresent study. Such decreases in tryptophan could readily leadto inhibition of protein synthesis and to low levels of serotonin,potentially causing neurological, vascular, and muscularchanges. Additionally, serotonin may be involved in release ofpituitary prolactin and growth hormone (37), in regulation of

interferon-induced la expression on macrophages (38), and inrelease or processing of messenger RNA from the cell nucleus(39). Thus, a variety of secondary effects may also result fromthis IL-2- and IFN-induced decrease in plasma tryptophan. Ifthe toxic or biological effects of systemically administered IL-2 or IFN-7 are, even in part, due to this induced tryptophandeficiency, then the provision of exogenous tryptophan (or arelated molecule such as 5-hydroxytryptophan, a serotoninprecursor) may potentially ameliorate some of these toxicities.On the other hand, if metabolites of tryptophan and KYN areresponsible for toxicity, especially when IDO levels are increased, as suggested by other data (40, 41), then supplementation with tryptophan may enhance toxicity and might not bedesirable. Determining the toxic versus protective effects oftryptophan or serotonin in animals receiving high doses of IL-2 or IFN-7 should resolve this issue.

Further testing of IL-2 is required before its clinical utility isdefined. Current data suggest its actions may be entirely via itsimmunopotentiating effects on lymphocytes, particularly a heterogeneous population of effector cells designated lymphokine-activated killer cells which includes subpopulations of T-cellsand natural killer cells. The potential clinical benefits of IL-2in cancer are believed to be based on its induction of antitumorimmunological activity. In this setting, the provision of exogenous tryptophan (or congeners) would not be expected to interfere with the desired effect of IL-2, could potentially amelioratesome of the toxic effects, and may enhance tumor cell killingby IFN-7-activated macrophages (42). However, if serotonin

plays a role in regulation of expression of la cell surface markers, as shown in mouse studies (38), then alterations of tryptophan metabolism may also play an indirect regulatory role inthe magnitude of the immune-mediated tumor destruction activated by IL-2.

The administration of IL-2 is known to induce secretion ofIFN-7 in vitro. High doses of IL-2, particularly by bolus administration, to patients with cancer have caused transient butdetectable levels of IFN-7 in the serum (43). It remains uncertain whether the toxicities of IL-2 and the alterations in tryptophan metabolism by IL-2 are, at least in part, due to thedirect effect of endogenous IFN-7 induced after IL-2 administration. Patients treated in this study did not have any life-threatening toxicity and the IL-2 at these doses was relativelywell tolerated, without any need for intensive care unit monitoring or support. IFN-7 levels were evaluated at multiple timesfor several of the patients receiving >106 units IL-2/nr/day but

at no time were significant elevations noted. However, in vitrostudies with human lymphocytes show potent induction of IDOby IL-2 which is inhibited by antiserum reactive against IFN-7(44). Thus, it seems most likely that the induction of IDO andNEO production are mediated, at least in part, via induction ofIFN-7 by IL-2. The antiproliferative effects of IFN-7 againstsome human cell lines in culture are apparently mediated byinduction of a tryptophan deficiency and are relieved by supplementation of media with tryptophan (45, 46). It, therefore,seems possible that at least some antitumor effects of IL-2 invivo may be the result of induced IFN-7, which may act indi

rectly via depletion of tryptophan.As IL-2 treatment trials have expanded, it is becoming in

creasingly important to identify how toxicity and immune activation relate to the potential for the antitumor response in anindividual patient. Cellular immune function studies /// vitrocan be performed reproducibly for single institution trials butare difficult to standardize for large multicenter cooperativetrials. In contrast, evaluation of tryptophan and NEO metabo-

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lism by analysis of plasma and/or urine is rapid and inexpensiveand can easily be performed on cryopreserved plasma and urinespecimens tested in a single reference laboratory. Further studies of these metabolic changes are required to determinewhether they may quantitatively correlate with immune activation, toxicity, or antitumor responses.

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1989;49:4941-4944. Cancer Res   Raymond R. Brown, Caroll M. Lee, Peter C. Kohler, et al.   Patients Treated with Recombinant Interleukin 2Altered Tryptophan and Neopterin Metabolism in Cancer

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