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[Cancer Biology & Therapy 1:2, 130-135, March 2002]; ©2002 Landes Bioscience

Research Article

Phase I Study of Perillyl Alcohol in Patients with Refractory Malignancies John R. Murren* Giuseppe Pizzorno Susan A. DiStasio Anne McKeon Kathleen Peccerillo Ashwin Gollerkari Walter McMurray Barbara A. Burtness Thomas Rutherford Xin Li Peter T. C. Ho Alan Sartorelli

ABSTRACT

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We treated 21 patients in a dose-finding and pharmacokinetic study of the monoterpene perillyl alcohol with the drug given orally in 3 divided doses on a chronic basis. The average number of days that patients remained on study was 48 (range 11-172). Fatigue and low-grade nausea were dose limiting. Using this schedule, a starting dose of 1.6 g/m2 with escalation to 2.1 g/m2 as tolerated is recommended. Two major metabolites were detectable and the mean peak plasma concentrations were 383 µM for perillic acid and 27 µM for dihydroperillic acid. The peak plasma concentration and the metabolite half-life were 2h and 1h post ingestion for perillic acid, and 4 h and 2.4 h for dihydroperillic acid, respectively. Stabilization of disease was observed in one of the 16 patients evaluable for response. Many of the gastrointestinal side effects that were poorly tolerated on a chronic basis may be partly related to the soybean oil base used in the current formulation. Further development of perillyl alcohol on this schedule would be facilitated by reformulation of the capsule.

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INTRODUCTION

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KEY WORDS

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Monoterpenes, Perillyl alcohol, Limonene, Dose-finding study

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This work was supported in part by U. S. Public Health Service grants (CA 16359, CA 75588, and CA 66739) from the National Cancer Institute, and from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.

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Previously published online as a CB&T "Paper in Press" at http://www.landesbioscience.com/journals/cancerbio/papersinpress/inpress12.html

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Received 8/28/01; Accepted 10/17/01

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*Correspondence to: John R. Murren, M.D.; Yale Cancer Center; Yale University School of Medicine; Section of Medical Oncology; 333 Cedar St., NSB 287; New Haven, Connecticut 06520 USA; Tel.: 203.737.1600; Fax: 203.785.7531; Email: [email protected]

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Monoterpenes are 10 carbon isoprenoids that are derived from the mevalonate pathway in plants, and are not produced by mammals.1 In citrus fruits, d-limonene (p-mentha-1,8-diene) is formed by cyclization of geranylpyrophosphate and then serves as a precursor for a number of other oxygenated monocyclic monoterpenes, including perillyl alcohol. These essential oils are largely responsible for the distinctive fragrance of citrus fruits, mint and herbs, and are frequently used as additives to food and cosmetics. In a number of animal models, monoterpenes have demonstrated activity against cancer both as chemopreventative and therapeutic agents. Dietary supplementation with the monoterpene limonene inhibits the development of chemically induced neoplasia in several rodent models, including the mammary gland,2,3 skin,4 and forestomach.5 In established rat mammary carcinomas, limonene produces complete regression in > 80% of the tumors.6,7 Perillyl alcohol is five-fold more potent than limonene in producing regression in both early8 and advanced mammary tumors,9 and also has activity in vivo against cancers of the pancreas,10 melanoma11 and liver12 and in vitro against neuroblastoma13 and colon carcinoma.14 The exact mechanism for the anticancer activity of monoterpenes has not been fully established. The chemopreventative effects may be related to induction of phase I and phase II enzymes resulting in carcinogen detoxification15,16 antiproliferative and/or pro-apoptotic activity, or may be due to induction of differentiation.7,9,12,17 Additional effects that may contribute to activity against established tumors include selective inhibition of prenylation of small GTP-binding proteins,14,18,19 modulation of other signal transduction pathways,12,20 or cell cycle blockade.13,21 Monoterpenes also alter mevalonate metabolism and some of these effects may contribute to the antitumor activity.22 Limonene reduces cholesterol biosynthesis through inhibition of 3-hydoxyl-3-methylglutaryl (HMG) coA reductase,23 whereas perillyl alcohol affects a target further downstream.18 Monoterpenes exhibit a high degree of oral bioavailability in mammals.24 Limonene is rapidly oxygenated to perillic acid and dihydroperillic acid, and these active metabolites are detectable in plasma within 20 minutes following administration by gavage in rodents.25 The metabolism of perillyl alcohol is similar to limonene in both rats and dogs.24 In single dose studies in beagles perillic acid and dihydroperillic acid were the only metabolites detected and represented 95% and 5% of the total area under the curve, respectively.24 Higher concentrations of monoterpenes are detected in adipose tissue and mammary gland than in less fatty tissue.25 Excretion occurs primarily through the urine.26 A dose-finding study of the monoterpene limonene was recently reported.27 The dose-limiting toxicities were nausea, vomiting and diarrhea. Although plasma concentrations

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Departments of Medicine, Pharmacology, Obstetrics and Gynecology and the Cancer Center, Yale University School of Medicine, New Haven, CT 06520

Cancer Biology & Therapy

2002; Vol. 1 Issue 2

PHASE I STUDY OF PERILLYL ALCOHOL IN PATIENTS WITH REFRACTORY MALIGNANCIES

comparable to the concentrations effective in vitro could not be achieved, intratumoral monoterpene concentrations were as much as 5.5 fold greater than the concentration achieved in plasma. Antitumor activity was observed. Perillyl alcohol has also entered clinical evaluation. Herein, we describe a dose-finding study of perillyl alcohol given chronically on a three-times-daily schedule.

PATIENTS AND METHODS Patients were eligible if they had histologically confirmed malignancies with measurable or evaluable disease for which no effective therapy was available. All patients were >18 years old, had an estimated life expectancy of at least 3 months, and had an Eastern Cooperative Oncology Group (ECOG) performance status 1500/µl, platelet count >100,000/µl, creatinine clearance >50 ml/min, SGOT grade 2 hematologic or >grade 1 non-hematologic drug-related toxicity were continued on treatment at one dose level below their current dose. Patients who experienced toxicity on the first dose level had the dose of perillyl alcohol reduced to 1.2 g/m2. Monitoring During Treatment, During the first 8 weeks of treatment the patients underwent weekly physical and toxicity assessments. In addition, weekly blood counts, liver function tests (SGOT, LDH, total bilirubin, alkaline phosphatase) and serum chemistries were obtained. In the event of grade 3 or 4 hematologic toxicity, follow-up blood counts were obtained every 2-3 days until there was evidence of hematologic recovery. For those patients who remained on treatment for greater than eight weeks, the above tests were obtained every 4 weeks. Formal tumor measurements of sentinel lesions were obtained after every 8 weeks of treatment. Pharmacokinetic Analysis. Heparinized blood samples were obtained on day 1 of treatment at 0, 1, 2, 4, 6, 8, 9, 17 and 25 hours following drug administration. Plasma specimens (100 µl) were combined with an aqueous solution consisting of 0.2 M phosphoric acid plus 1.0 M potassium chloride (100 µl) and TBME-IS (tributylmethylether-internal standard) solution (200 µl) in a 1.5 ml polypropylene microcentrifuge tube. Extraction by vigorously mixing on a vortex action stirrer for 1 min was followed by centrifugation (12000 x g. 10 min). The organic phase was extracted and evaporated to dryness. Perillyl alcohol and its major metabolites were measured according to the methods of Phillips et al24 with the following modifications: the internal standard concentration used was 5 µg/200 µl rather than 5 µg/ml, the metabolites were silylated with N,O- bis(trimethylsilyl)trifluoroacetimide (BSTFA) with 1% trimethylchlorosilane, and the mass spectra was acquired using ammonia chemical ionization. The internal standard was 5-methoxysalicylic acid (Aldrich). Samples were run on a HP5989B mass spectrometer and a HP5890 gas chromatograph equipped with an autosampler. The limit of detection was 2.0 µM for perillic acid and 1.5 µM for dihydroperillic acid. The assay was linear up to 750 µM for both metabolites with an interday variation of the assay averaging 8% and 12% for perillic acid and dihydroperillic acid, respectively. Data were analyzed using PC-NONLIN software (Scientific Consulting, Lexington, KY). The area under the curve (AUC) was determined using the linear trapezoidal rule.

RESULTS Patient Characteristics. Twenty-one patients were entered on the study. All of the patients had a performance status of 0 or 1 as defined by the Eastern Cooperative Oncology Group (ECOG) and had received prior cytotoxic chemotherapy (Table 1). The median number of previous chemotherapy regimens was 3 (range 1-6), and 11 patients had received prior radiation therapy. One patient had been previously treated with intensive chemotherapy and peripheral blood stem cell transplant. Patients were enrolled on three dose levels of perillyl alcohol (Table 2). DLT consisted of fatigue and occurred in all patients treated on the third dose level of 2.8 g/m2. The average number of days that patients remained on study was 48 (range 11-172). Sixteen patients were evaluable for response. In fifteen of these patients treatment was discontinued because of progressive disease at a median of 52 days. There was 1 patient who experienced stable disease for a duration of 169 days. Five patients were not evaluable because of early withdrawal due to unacceptable side effects. A patient on the first dose level developed a near constant oily rectal seepage on day 41 of therapy and refused further therapy. This symptom resolved 20 days later. Three of the patients on the second dose level of 2.1 g/m2 discontinued treatment


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Table 1

study. Four of the eighteen patients that received 2.1 g/m2 also developed severe fatigue during treatment. Although progression of the underlying cancer contributed to the fatigue, notable improvement in the symptom occurred in several patients within 3 days of discontinuing the perillyl alcohol. There was no correlation between the incidence of fatigue and baseline performance status or the number of prior chemotherapy regimens. Every patient experienced mild-to-moderate gastrointestinal toxicities that included nausea, diarrhea, bloating, and anorexia. In some patients premedication with compazine or ingestion of a light meal before taking the pills partially ameliorated the nausea. Nausea was significant in four patients but in only one of these patients was the symptom tolerable when the dose was reduced. Occasional vomiting (grade 1) accompanied the nausea in 10 patients, and was observed with equal frequency on each dose level. Despite near universal complaints of some degree of nausea and anorexia, only 3 patients, all of whom were enrolled on the second dose level, experienced weight loss exceeding 5% of their baseline weight and no patients experienced weight loss exceeding 10% of baseline. Asymptomatic grade 1-2 transaminitis developed in three patients. All of these patients were treated on the second dose level and all had known liver metastases. The serum chemistries were suggestive of hepatocellular damage without an element of cholestasis. In two of the patients the transaminases began to rise within 2 weeks of starting treatment, and in the other patient the transaminases increased after 56 of therapy and coincided with radiographic evidence of progressing liver metastases. Central nervous system toxicities, manifested as mild disorientation, loss of balance, and impaired ability to concentrate were observed in 11 patients. One patient on the third dose level also developed slurred speech. These symptoms appeared to be dose-related and resolved with withdrawal of the drug. Some patients were treated at a lower dose without recurrence of this toxicity. All of the patients were also taking antiemetics which may have contributed to the observed side effects. Dose Modifications. Dose modifications were necessary in all three patients treated on the third dose level (2.8 g/m2) due to fatigue. None of these 3 patients received more than 10 days of treatment before a dose reduction was made. Two of the 3 patients were able to tolerate treatment on the second dose level but the third withdrew from the study within 2 weeks of her first dose of perillyl alcohol. Seven of the 15 patients that started treatment on the second dose level of 2.1 gm/m2 had dose reductions because of toxicity. Dose modifications were necessary because of unacceptable gastrointestinal side effects (3), fatigue (3) and CNS toxicities in 1 patient, respectively. Perillyl alcohol was not well tolerated even at a lower dose in four of these seven patients, and three withdrew from the study because of toxicity. None of the patients who were treated on the first dose level required dose modifications. Duration of Treatment. All 3 patients on the first dose level (1.6 gm/m2) completed at least 4 weeks of treatment (range 42-172 days) before documentation of disease progression. Seven of the 15 patients that started

PATIENT CHARACTERISTICS

Enrolled

21

Sex Male Female

9 12

Age, years Median Range

55 40-79

Performance Status 0 1

5 16

Tumor Histology Pancreas GI Ovarian NSCLC Breast Other*

2 6 2 2 3 6

Prior therapy Chemotherapy Radiation therapy

21 11

*Salivary gland (3), adrenal (1), cervical (1), sarcoma (1)

because of toxicity despite attempts at dose modification. One patient who was treated on the third dose level discontinued therapy after 12 days of treatment because of unacceptable fatigue. Hematologic Toxicity. Hematologic toxicity was modest. Grade 2 or 3 anemia was recorded in four patients, but all had baseline hemoglobin 2.1 g/m2 during the 14 days they ingested the drug.36 All of the studies to date have attempted to deliver monoterpenes on a continuous basis, since this drug appears to be primarily a cytostatic agent and this was the schedule that was successfully tested in animals. However, although peak plasma concentrations of the perillyl alcohol metabolites in our trial and in the other reported studies were above the concentration that produced tumor regression in a rodent model,7,9 we saw no evidence of tumor regression, and only one patient with metastatic adrenocortical carcinoma had disease stabilization that lasted 5 months. Activity with other schedules of perillyl alcohol has been similarly modest. One patient treated at a dose of 1.6 g/m2 four-times-daily had near complete response of multiple pulmonary lesions due to chemotherapy-resistant colon cancer.35 Stabilization of disease has been noted in a few other patients with colon cancer and in a few patients with hormone refractory prostate cancer.28,35,36 Even if tumor cytostasis were achieved, chronic administration of perillyl alcohol would be difficult because of the significant impact on quality of life. At sufficiently high concentrations (~5 mM), 24 h exposure to perillyl alcohol is cytotoxic to cultured cells.19 This cytotoxic concentration is 7-fold higher than the maximum concentration of monoterpenes achieved in plasma in the studies reported thus far. However, with intensive premedication a once weekly or once every 2-week schedule of dose-intensified perillyl alcohol could be explored and may be better tolerated by patients. This approach would require a reformulation so that the number of capsules ingested was reduced. Reformulation of the capsule, in which the ratio of perillyl alcohol to soybean oil is increased, is being considered and would reduce both the number of pills required and the fat load ingested by patients. This would result in better patient acceptance of this therapy and may permit greater dose escalation of the perillyl alcohol on either a chronic or intermittent schedule. Analogs that are more potent inhibitors of prenylation are also under development, and may provide useful alternatives.40 Ongoing studies should define whether there is a role for chronic administration schedules of the current formulation of perillyl alcohol. Whether or not perillyl alcohol would be useful as a chemopreventative agent in humans is not known. Although it successfully inhibits the induction phase of carcinogenesis in several animal models,2-5,8,12,15,16 it promoted tumorogenesis in at least one rat hepatic cancer model,41 making it unlikely to be administered to healthy patients until more efficacy data is established in humans. Even if the ongoing studies of perillyl alcohol in established cancers are unsuccessful, further clinical development of more potent derivatives of this interesting class of compounds is warranted. References 1. Chayet L, Rojas C, Cardemil E, Jabalquinto AM, Vicuna R, Cori O. Biosynthesis of monoterpene hydrocarbons from [1-3H]neryl pyrophosphate and [1-3H]geranyl pyrophosphate by soluble enzymes from Citrus limonum. Arch Biochem Biophys 1977; 180(2):318-27. 2. Elson CE, Maltzman TH, Boston JL, Tanner MA, Gould MN. Anti-carcinogenic activity of d-limonene during the initiation and promotion/progression stages of DMBA-induced rat mammary carcinogenesis. Carcinogenesis 1988; 9(2):331-2. 3. Maltzman TH, Hurt LM, Elson CE, Tanner MA, Gould MN. The prevention of nitrosomethylurea-induced mammary tumors by d- limonene and orange oil. Carcinogenesis 1989; 10(4):781-3.


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