4-Hydroxyphenylretinamide in the Chemoprevention of Cancer

Harmesh R. Naik* Gregory Kalemkerian* Kenneth J. Pientat

* Meyer L. Prentis Comprehensive Cancer Center

Wayne State University School of Medicine Division of Hematology and Oncology Detroit, Michigan 48201

t University of Michigan Comprehensive Cancer Center

University of Michigan School of Medicine Division of Hematology and Oncology Ann Arbor, Michigan 48 I09

4-Hydroxyphenylretinamide in the Chemoprevention of Cancer

1. Introduction Vitamin A plays a very important role in maintaining normal vision and promoting growth and differentiation of normal epithelial tissues (Bollag and Matter, 1981). In numerous experimental investigations vitamin A and its analogs have demonstrated activity in modulating growth and cellular maturation and differentiation in many cell types (Lotan, 1993). Vitamin A analogs have shown promising activity as both preventive and therapeutic agents against malignancy in experimental models (Bollag and Peck, 1993); however, use in humans has been restricted because of toxicity to the liver, the predominant storage site for most retinoids. The search for less-toxic and more-active retinoids has led to the synthesis of numerous structurally modified analogs, and N-4-hydroxyphenylretinamide(4HPR or fenretinide) is one of the new synthetic retinoids which has been found to be effective and less toxic than vitamin A itself. In experimental chemoprevention studAdvances in Pharmacology, Volume 33 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved

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ies, 4HPR has been shown to reduce carcinogen-induced cancers and several human trials are currently ongoing utilizing this agent (Costa, 1993). In this review we will summarize the current status of 4HPR as a chemoprevention agent in cancer.

II. Chemoprevention Concepts Chemoprevention is a term used to describe a novel approach of reducing risk of development of cancer in susceptible individuals by administration of chemical compounds which have the potential to reverse or suppress carcinogenesis (Sporn and Newton, 1979; Sporn and Roberts, 1984). Since most cancers are incurable with any available treatment modality at present, prevention of cancer development is an appealing health care strategy. To achieve successful chemoprevention, an understanding of the carcinogenic process as well as effective chemical compounds are required. In the last few years significant advances have been made in understanding that carcinogenesis is a multistep process involving initiation, promotion, progression, neoangiogenesis, and metastasis (Szarka et al., 1994). Chemopreventive agents can be categorized into four subtypes based on the stage of carcinogenesis at which a compound acts (De Palo, 1992; Boone et al., 1990). (1) Inhibitors prevent the onset of carcinogenesis from precursors (antiinitiation). (2) Blocking agents prevent the carcinogen from reaching or reacting with the target cell (anti-initiation, antipromotion). (3) Suppressive agonists prevent neoplastic transformation in cells that are exposed to a carcinogen by inducing or enhancing differentiation (antipromotion, antiprogression, e.g., retinoids). (4) Competing agents block the proliferative stimulus by competitively inhibiting receptors (antipromotion, antiprogression, e.g., tamoxifen).

Retinoids are the most commonly used chemoprevention agents but their mechanism of chemopreventive action is not fully understood. It is hypothesized that retinoids modulate gene expression via interaction with nuclear retinoic acid receptors, thereby affecting cellular differentiation and suppressing progression of preneoplastic cells to frank neoplastic lesions (Lotan, 1993). Proliferation inhibition by retinoids may be related to induction of the terminal differentiation process; however, a direct antiproliferative effect is also believed to play an important role (Bollag and Peck, 1993).

111. Retinoids Retinoid is a generic term used to describe vitamin A or related compounds, including retinol and other natural or synthetic analogs. The plant

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pigment p-carotene, present in various fruits and vegetables, is a major source for vitamin A and is the most active of the plant carotenes. Butter, cheese, liver, fish, and egg yolk are other significant sources of dietary vitamin A. The natural retinoids exist as alcohols, aldehydes, acids, and esters, and exist in all-truns, 13-cis, or 1 1-cis configurations (Blaner, 1993). Retinol, the primary alcohol, is present in esterified form mainly in the liver. Retinol exists in trans and cis isomeric forms which can be interconverted in the body and serves as a precursor for retinyl ester (which is the storage form), retinaldehyde, and retinoic acid. All-trans-retinoic acid appears to be the active form of vitamin A in all tissues except the retina, where the 11-cis form of retinaldehyde plays a central role in maintaining vision (Blaner, 1993; Marcus and Coulston, 1993).

A. Structure The basic structure of a retinoid consists of a beta ionone cyclic ring, which is required for activity, a polyene side chain, and a polar end unit (Figure 1).Modifications in this basic structure have produced more than 1000 retinoids with differing metabolism, storage, transport, biologic activity, and toxicity. In second generation retinoids the p-ionone ring is aromatized (e.g., acitretin). The third generation retinoids, arotinoids, feature two

all-trans-retinoic acid

13-cis-retinoic acid

4-hydroxyphenyl retinamide

FIGURE I Structure of different retinoids.

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aromatic rings and are highly potent. In humans, all-trans-retinol is the standard compound against which the activity of the synthetic retinoids is compared.

B. Physiology of Retinoids Different retinoids and their analogs may have different mechanisms for transport and metabolism depending on their structural differences. Alltruns-retinoic acid, 13-cis-retinoic acid, retinamide derivatives, and aromatic analogs have all been studied in detail (Blaner, 1993; Kalin et al., 1981, 1982). 1. Dietary Intake: Intestinal Absorption of Retinoids

Vitamin A is a fat soluble, essential vitamin which cannot be synthesized in humans and must be ingested in the diet to maintain physiologic levels. Retinoids contained in the diet, either as /3-carotene (in fruits and vegetables) or preexisting retinoids (in milk, butter, and fish), are absorbed from the gastrointestinal tract. p-Carotene can either be converted into retinol in the intestinal lumen or directly absorbed from the small intestine unchanged (Szarka et al., 1994). In the intestinal mucosa, /3-carotene can be cleaved to retinaldehyde (Lakshman et al., 1989). Dietary retinoids are absorbed after being converted into retinol. Retinol uptake by intestinal cells is carrier mediated and facilitated by a high-affinity cytosolic protein, cellular retinol binding protein I1 (CRBP 11). CRBP I1 is localized solely in small intestine and, in addition to retinol, can also bind to retinaldehyde. When bound to CRBP 11, retinaldehyde, which is a cleavage product of carotene, is reduced to retinol by the mucosal enzyme retinaldehyde reductase (Kakkad and Ong, 1988). Retinol is then esterified in intestinal cells by lecithin retinol acyltransferase (LFUT) and packaged as retinyl esters into chylomicrons (Quick and Ong, 1990). Thus, retinaldehyde formed from dietary carotene or directly absorbed retinol is metabolically channelled through binding to CRBP I1 for packaging into chylomicrons as retinyl ester. The chylomicrons are secreted into lymphatics and catalyzed by lipoprotein lipase to give rise to chylomicron remnants. 2. Hepatic Uptake and Storage

Chylomicron remnants are taken up by liver parenchymal cells through a cell-surface receptor which recognizes the apolipoprotein components of the chylomicron remnants (Mahley and Hussain, 1991). The parenchymal cells first hydrolyze retinyl ester to retinol and then transfer it to stellate cells, specialized cells for retinoid storage and metabolism. The exact mechanism of transfer of retinol from liver parenchymal cells to stellate cells is not known. More than 75% of hepatic retinoid is stored in stellate cells as retinyl

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ester in the form of lipid droplets. The stellate cells contain CRBP (cellular retinol binding protein), CRABP (cellular retinoic acid binding protein), and the enzymes retinyl ester hydrolase, which converts retinyl ester to retinol, and LRAT, which catalyzes the formation of retinyl esters. For maximum activity LRAT requires retinol bound to CRBP (Blaner, 1993). Some of the dietary retinoid internalized by liver parenchymal cells can be directly secreted into the circulation bound to retinol binding protein (RBP) which is synthesized by liver parenchymal cells. The relative proportions of dietary retinoid secreted into the circulation or channeled to stellate cells for storage depends upon the retinoid status of the animal. In a lowretinoid intake state, a smaller percentage of the dietary retinoid will be transferred for storage (Batres and Olson, 1987). Adipose tissue also contributes significantly to retinoid storage. Approximately two-thirds of the retinoid present in adipose tissue is in the retinol form; the remainder is stored as retinyl esters (Blaner, 1993). 3. Transport in Circulation

Prior to entry into the circulation from the liver, hepatic retinyl esters are hydrolyzed to retinol. In the circulation retinol is 95% bound to the specific transport protein RBP. RBP is complexed with another protein called transthyretin, which is thought to protect RBP from renal excretion. Retinol, in part, is conjugated to form a p-glucuronide which undergoes extrahepatic circulation and is oxidized to retinal and retinoic acid. Several water soluble metabolites are excreted in urine and feces (Marcus and Coulston, 1993). 4. Target Cell Uptake

Retinol is delivered to the target tissues as retinol-RBP complex. The uptake mechanism by the target cell is an area of controversy. Some studies suggest the presence of specific cell surface receptors which recognize RBP (Sivaprasadarao and Findlay, 1988a,b), while other studies using model membrane systems have suggested that uptake of retinol does not require cell-surface receptors for RBP. In the target cells, retinol is believed to undergo two oxidation reactions, first to retinaldehyde and then to retinoic acid (Blaner, 1993). The retinoic acid is conveyed to the nuclear receptors bound to CRABP. In the retina, retinol is converted to 11-cis-retinal, which combines with opsin to form rhodopsin. On exposure to light 11-cis-retinal is converted to all-trans-retinal and opsin is dissociated. All-trans-retinal can be reduced to all-trans-retinol, which is then converted to 11-cis-retinol, which can be recycled (Marcus and Coulston, 1993). 5. Interaction at the Receptor Level

Retinoic receptors are targets of intense study to understand molecular mechanisms of retinoid action. Two classes of retinoid binding proteins have been reported, one cytoplasmic and the other nuclear. Four cytoplasmic

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proteins have been identified. Two cytoplasmic retinol binding proteins (CRBP I and 11) bind to retinol, while the other two bind to retinoic acid and are called cellular retinoic acid binding proteins (CRABP I and 11). These binding proteins appear to be involved in retinoid transport and metabolism within cells. The nuclear retinoic acid receptors (RAR) appear to be the most important mediators of retinoid actions. RARa, RARP, and RARy act as ligand-dependant transcription factors, which bind to specific DNA sequences. When retinoic acid binds to the receptor, a conformational change in the RAR occurs followed by formation of transcriptional initiation complexes. In most instances retinoic acid induces gene transciption, but inhibition of transcription can also occur. Recently a new family of receptors, named RXRa, RXRP, and RXRy, has been identified. Although retinoic acid does not bind to the RXRs directly, it is believed to induce RXRdependant transcription activation via one of its metabolites, 9-cis-retinoic acid. The retinoic acid receptors have been reviewed elsewhere in detail (McBurney et al., 1993).

IV. 4HPR Pharmacology The pharmacological profile of 4HPR differs markedly from that of other retinoids. The main difference is the absence of hepatic accumulation of 4HPR, which reduces liver toxicity, and selective uptake by certain tissues, such as mammary tissue, which makes it an attractive agent for chemoprevention of breast cancer.

A. Animal Data 1. Pharmacokinetics Early studies in rats after oral administration of 4HPR indicated selective accumulation of 4HPR and its metabolites in the mammary glands in a dose-dependant manner, without any detectable rise in hepatic retinoid levels (Moon et al., 1979). Even after 6 months of 4HPR administration there was no elevation of liver retinoid levels. This favorable distribution resulted in a significant reduction in observed toxicity during chronic administration of 4HPR. The pharmacokinetics of 4HPR have been studied in rats after intravenous and oral administration (Swanson et al., 1980). After a single 5 mg/kg intravenous dose, a triexponential plasma concentration curve was obtained. The volume of distribution was found to be 10-12 liters/kg and terminal plasma half-life was 12 hr, significantly longer than its natural analog, all-trans-retinoic acid (20 min). Tissue distribution data from the same study confirmed that, even though hepatic levels of 4HPR were tran-

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siently elevated after each dose, 4HPR and its metabolites are not stored in the liver. This was an important observation since hepatic accumulation and subsequent toxicity precludes prolonged administration of many other retinoids. In addition, 4HPR was retained much longer in adipose tissue than in other tissues, as indicated by a higher tissue/plasma (TIP)ratio. High TIP ratios seen for stomach and large and small intestines were the result of direct exposure to the drug. Less than 2 % of the administered 4HPR was excreted unchanged, suggesting the importance of biotransformation in the elimination of 4HPR from the body. After an oral dose of 10 mg/kg of 4HPR, the peak concentration was achieved in 4 hr, but the plasma levels were lower than those seen after intravenous administration. In addition, the elimination half-life of oral 4HPR was shorter than that seen after intravenous administration. These data suggest incomplete absorption through the oral route (Swanson et al., 1980). Further studies on biotransformation indicated extensive metabolism of 4HPR to a lipophilic compound, 0-methylHPR (MPR), and numerous polar retinamides, including HPR 0-glucuronide (Swanson et al., 1981). Polar metabolites are excreted into urine and bile, while the nonpolar metabolite (MPR) tends to accumulate into tissues such as fat, prostate, liver, skeletal muscles, and intestines. The biologic activity of MPR is equivalent to that of 4HPR in in vitro experiments. The half-life of MPR (26 hr) is twice that of 4HPR. The longer half-life and increased lipophilicity explain the selective retention of MPR. From this information the authors concluded that MPR is likely to contribute significantly to the overall pharmacological effects of 4HPR in vivo. An alternative metabolic pathway involving the hydrolysis of 4HPR by amidases to release retinoic acid has been suggested, but no definite experimental evidence was found to support this hypothesis (Swanson et al., 1981). Another study investigated the distribution and metabolism of 4HPR in female mice after a single oral dose of 10 mg/kg (Hutlin et al., 1990). The half-life values for MPR, the major metabolite of 4HPR, were reported to be 5.1 hr in liver, 5.6 hr in serum, 18.7 hr in bladder, 23.1 hr in skin, and 26.6 hr in mammary tissue. The highest levels of MPR were detected in liver and mammary tissue. Two studies examining the pharmacokinetics of the sister compound N-2-(hydroxyethyl)retinamide(HOERA) in mice have been reported. Following an intravenous dose of 10 mg/kg of HOERA, a distribution phase lasting 1 hr was followed by an exponential phase of elimination, which was slower than that of 13-cis-retinoic acid (Wang et al., 1980). At 18 hr, considerable concentrations of HOERA were still found in liver, kidney, and testes, indicating accumulation of the drug in these tissues. No cleavage from HOERA to retinoic acid was found in examined tissues. After an oral dose of 10 mg/kg of HOERA, the maximum serum concentration was reached in 15 to 30 rnin and declined exponentially with a half-life of

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2.9 hr. The tissue concentration increased to maximum in 30 min to 2 hr, except in small intestine where rapid accumulation occurred (time to highest concentration being 5 min). HOERA disappeared from tissues exponentially with half-life values ranging from 2.1 to 4.7 hr. In the bladder, the disappearance was delayed with a half-life of 7.3 hr. Oral administration also resulted in lower bioavailability (60%)when compared to intravenous administration. HOERA again appeared to be concentrated in tissues at higher levels than in serum. Small amounts of unchanged drug were reported in urine, feces, and bile (Kalin et al., 1982). These observations were comparable to results from 4HPR pharmacokinetic studies (Swanson et al., 1980). In summary, it is evident from animal pharmacokinetic studies that 4HPR exhibits a longer half-life than many other retinoids, achieves higher tissue concentrations relative to plasma, has selective accumulation in certain tissues (e.g., breast), and appears to have active metabolites. Most importantly, 4HPR and its metabolites are not stored in the liver, making chronic administration possible without significant hepatotoxicity.

B. Human Data 1. Bioavailability and Diet Composition The effect of meals and meal composition on the bioavailability of 4HPR in healthy male volunteers has been reported in a recent study by Doose et al. (1992). After a single fasting oral dose of 300 mg in capsule form, peak plasma concentrations for 4HPR and MPR were 198 ng/ml and 91 ng/ml with time to peak concentration being 5.2 and 7.7 hr, respectively. When the same dose was administered after a meal, the peak concentrations of 4HPR and MPR were increased 3 and 2.3 times, respectively, and the peak was delayed. The AUC (area under the curve) values also showed a threefold increase with meal administration. No clinically significant adverse reactions or changes in the laboratory values were reported. Further study of the effect of food composition revealed that 4HPR administration after a high-fat meal resulted in the greatest bioavailability, highest peak concentration, and longest time to peak when compared to high-protein and highcarbohydrate meals. The high-protein meal resulted in a modest increase in bioavailability compared to the high-carbohydrate meal (Doose et al., 1992). The delay in time to peak concentration after a fatty meal can be explained by more extensive lymphatic uptake secondary to increased chylomicron formation after a high-fat meal. Improved absorption after a protein meal may be indicative of a hemodynamic mechanism such as increased splanchnic blood flow which has been shown to correlate with protein quantity in a given meal.

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2. Pharrnacokinetics

The pharmacokinetics of 4HPR and its major metabolite, MPR, was evaluated in cancer patients entered into a phase I1 clinical trial using oral 4HPR at a dose of 300 mg/m2 daily (Peng et al., 1989). A sensitive HPLC assay was utilized to measure the retinol, 4HPR, and MPR levels. The average plasma terminal-phase half-lives for 4HPR and MPR were 13.7 and 23 hr, respectively. The average total body clearance rates for 4HPR and MPR were 56.6 and 239.3 liters/hr/m2, respectively. The plasma AUC for 4HPR was twice that of MPR. After a single oral dose the peak plasma concentration was 509 ng/ml for 4HPR and 80 ng/ml for MPR. The time to peak plasma concentration was between 3 and 4 hr for 4HPR and 8 and 12 hr for MPR. In an Italian study, 4HPR and MPR levels were monitored in a group of patients who were participating in a phase I trial (Formelli et al., 1989). Patients received 100, 200, and 300 mg 4HPR or placebo orally every day for 6 months and then all patients received 200 mg 4HPR for 6 more months. At 5 months, there was a linear correlation between dose and 4HPR and MPR plasma levels 12 hr after the last dose. The plasma levels were repeated at 9 and 12 months in each group to determine if any accumulation occurred. In the group initially receiving 300 mg of 4HPR followed by 200 mg, 4HPR and MPR levels remained high at 12 months, suggesting accumulation of 4HPR. In the group receiving the 200-mg dose, 4HPR levels remained unchanged, suggesting no drug accumulation. After drug interruption, the rate of decrease of plasma levels was slower for MPR than for 4HPR. After 50 days, a very low amount of 4HPR was detected in plasma but MPR was still present at a higher concentration than that of 4HPR. These observations are important since prolonged administration of a retinoid could lead to accumulation of drug and significant toxicity. 4HPR’s plasma and breast tissue kinetics were recently reported in humans. Fourteen women scheduled to have surgery for breast abnormalities were given 4HPR orally at 300, 200, or 100 mg/day for 3-7 days prior to surgery. Plasma and breast tissue (normal and neoplastic) levels were determined. There was a dose-dependant uptake of 4HPR and MPR in normal and neoplastic breast tissue from plasma with a 10 : 1 breast : plasma concentration ratio, suggesting concentration of 4HPR in breast tissue (Modiano et al., 1993). These results confirm previous animal data indicating selective concentration of 4HPR in mammary tissue. In another study, levels of 4HPR and its metabolites were measured in breast tissue obtained a t surgery. (Mehta et al., 1991). Two metabolites were detected in tissue extracts, MPR and a yet-unidentified metabolite. The concentrations of both 4HPR and MPR were higher in the breast tissue than in plasma and the breast tissue MPR concentrations were always higher than the 4HPR concentrations. Interestingly, 4HPR localized to breast epi-

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thelial cells in contrast to MPR, which was concentrated in the fat compartment, indicating selective distribution. At present, it is not known if MPR can be metabolized back to 4HPR in breast tissue, as has been observed for the mouse mammary gland in vitro (Mehta et al., 1988b).

V. Effect of 4HPR on Vitamin A Patients taking 4HPR sometimes develop adverse effects similar to those seen in patients with vitamin A deficiency, including impaired dark adaptation. Since dietary insufficiency is not suspected in these patients, interference with normal retinol metabolism has been suggested as a likely explanation for these symptoms. The pharmacological basis for this reasoning was explored in the following studies.

A. Studies in Animals In a study investigating the effect of a diet containing 4HPR on retinol metabolism in normal and vitamin A-deficient rats, serum retinol levels were significantly lower in 4HPR-treated animals (Schaffer et af., 1993). On Day 2, serum retinol levels decreased by 50% in 4HPR-treated rats compared to pretreatment levels and were 52% lower than in the control group. O n Day 14, serum retinol levels were reduced to 42% of both pretreatment and control groups. Serum RBP levels were lower in 4HPR-treated animals, whereas other proteins did not show significant differences between the control and 4HPR groups. On Day 2 of 4HPR treatment, rats showed a significant 54% reduction in serum RBP concentration compared to pretreatment concentration, and the values were 57% lower than in control rats. Serum RBP concentrations remained low over the entire 14-day period in 4HPR-treated normal rats. In addition, liver and kidney RBP concentrations were higher in the 4HPR-treated group than in controls. In vitamin A-deficient rats, retinol repletion caused a large increase in serum RBP after 30 min, while in the 4HPR-treated group, serum RBP levels decreased at 30 min and remained low thereafter (4.06 vs 0.34 pmol/liter a t 150 min in the retinol-repleted vs 4HPR group, respectively). The 4HPR group also had lower liver RBP levels but higher kidney RBP levels than the control group. The authors concluded that 4HPR induces RBP secretion from the liver into the blood stream, from which it rapidly accumulates in the kidneys. There is some evidence that 4HPR may interfere with the absorption of vitamin A. 4HPR inhibited retinol esterification by intestinal and liver LRAT, presumably by interaction with the binding site of the enzyme (Dew et al., 1993). Solubilization resulted in further inhibition, thus suggesting enzyme inhibition in addition to a membrane effect. 4HPR also inhibited

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intestinal retinal reductase activity in a concentration-dependant manner, suggesting that these interactions may significantly interfere with the intestinal processing of vitamin A and may work in concert with the effects of 4HPR on U P .

B. Human Data The effect of 4HPR on vitamin A has been examined in four clinical trials. A rapid and significant decrease in plasma retinol and RBP levels was seen after oral 4HPR intake in patients on a phase I1 study, with the mean plasma retinol concentration for nine patients taking 4HPR decreased by 60% from baseline within 1-2 weeks of 4HPR initiation (Peng et al., 1989). The rapidity of decrease in plasma retinol and RBP levels suggested mechanisms other than deficient vitamin A stores since plasma retinol concentrations can be maintained at normal levels over many months at the expense of hepatic reserves even with ingestion of a vitamin A-free diet. Addition of 4HPR to pooled human plasma in vitro did not change plasma retinol or RBP levels, which ruled out a direct chemical interaction. A phase I study from Italy reported similar declines in retinol levels after 5 months of treatment with three different doses of 4HPR (100, 200, or 300 mg). Retinol levels were significantly lower in all 4HPR-treated patients than in patients receiving placebo, and a negative linear relation was found between the dose of 4HPR and retinol levels (Formelli et al., 1989). Within 24 hr of the first dose of 4HPR (200 mg), retinol and RBP concentrations were reduced by 38 and 26%, respectively. After the initial 6 months, all patients received a similar dose of 4HPR (200 mg) for another 6 months. No further reduction in retinol levels was noted in patients taking the 200-mg dose. When 4HPR therapy was interrupted, the retinol levels returned to the range of those of the placebo group, suggesting reversibility of this effect. The exact significance of the reduction in retinol level is not known; however, it may explain the visual problems encountered during HPR administration and rapid reversal of symptoms on discontinuation of HPR. Furthermore, no information is available on the relevance of lowretinol levels to the chemopreventive activity of 4HPR. In a third study, 4HPR was given to women 3-7 days prior to breast surgery and plasma, and breast tissue retinol levels were measured. 4HPR reduced retinol levels by 32-42% in plasma and 40-57% in breast tissue in a dose-dependant manner (Modiano et a/., 1993). The fourth study reported similar results. In this trial patients received 200 mg/day of 4HPR for 1 year after surgical treatment of oral leukoplakia. After 1 year, plasma retinol levels were significantly reduced from baseline in the 4HPR group. In addition, plasma retinol levels at 1 year were lower in the 4HPR group as compared to levels in the control group (Chiesa et al., 1992).

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VI. Experimental Data on 4HPR as a Chemopreventive Agent Retinoids have been shown to control cellular differentiation and proliferation in most epithelial tissues and, therefore, have been proposed as chemopreventive agents (Sporn et al., 1976). 4HPR preferentially accumulates in mammary tissue, thereby providing a unique rationale for studying its ability to prevent breast cancer (Sporn and Newton, 1979).

A. In Vitro Systems An extensive literature exists on effects of retinoids on cellular growth and maturation of various cell types (Bollag and Peck, 1993; Moon, 1993; Lotan, 1993). For this review we will limit the discussion to data available on 4HPR. I. Effects on Differentia tion The effect of 4HPR on epithelial cell differentiation was first demonstrated in hamster tracheal organ cultures. In this system, vitamin A deficiency results in keratinizing squamous metaplasia. At concentrations of lo-* M, 4HPR completely reversed keratinization and was more potent than retinyl acetate (Moon et al., 1979). In another study, 4HPR was among the most potent of 87 retinoids tested in reversing keratinization of hamster tracheal organ cultures (Newton et al., 1980). 2. Effeck on Proliferation The antiproliferative activity of 4HPR was reported to be 10-fold higher than that of retinoic acid (RA) against the T24 human bladder cell line (McCormick et al., 1985). Proliferation of three more human bladder carcinoma cell lines, EJ, 582, and SCaBER, was also inhibited by 4HPR and each of these four cell lines metabolized 4HPR to RA. In in vivo experiments, bladder contained the highest [3H]RA concentrations 3, 6, and 12 hr after oral [3H]4HPR administration to normal and 4HPR-treated rats. Rat bladder homogenates contained retinamide hydrolase activity which converted 4HPR to RA in vitro. The authors concluded that combined in vivo and in vitro metabolism data suggested conversion of 4HPR to RA in rat bladder, and RA accumulation could be responsible for chemopreventive activity of 4HPR. However, these findings cannot explain the 10-fold greater activity of 4HPR. In contradiction to above results, another in vitro study using rat and human bladder cancer cell lines showed growth inhibition in a dosedependant and reversible manner by all-trans-retinoic acid, while 4HPR was found to be ineffective in this system (Jones et al., 1989). 4HPR also inhibited proliferation of cultured human breast cells (Marth et al., 1985) and human stem cells from ovary, lung, and melanoma

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(Meyskens et al., 1983). 4HPR was found to inhibit the growth of rat and human prostate cancer cells as well as bovine pulmonary artery endothelial cells in vitro (Pienta et al., 1993). 4HPR inhibited the growth of several lymphoid and myeloid malignant lines in a dose-dependant manner (Delia et al., 1993). Interestingly, the RAresistant leukemic cell lines HL-60R and NB-306 were highly susceptible to 4HPR, suggesting that these two retinoids may act through different mechanisms of action. The effects of 4HPR on proliferation and differentiation, however, are not universal. In the human myeloid leukemia cell lines HL-60 and KG-l,4HPR did not show any antiproliferative or differentiating activity, while all-trans-retinoic acid was highly active (Tobler et al., 1986). 3. Effects on Carcinogenesis

Bertram and colleagues (1981) determined that 4HPR was the most potent of the retinoids tested in inhibiting carcinogen-induced transformation in the mouse embryo fibroblast 10T1/2 cell line. 4. Other Effects

Retinoids have been demonstrated to inhibit angiogenesis (Oikawa et al., 1989). At a concentration of 2.5 p M , 4HPR inhibited angiogenesis, endothelial cell motility, and tubule formation in vitro (Pienta et al., 1993). 4HPR has been found to induce apoptosis in the lymphoma cell line DoHH2 at a time when no differentiation is evident (Delia et al., 1993). In summary, 4HPR has been demonstrated to have antiproliferative, antiangiogenic, cytotoxic, and differentiating activity in in vitro studies.

B. In Vivo Systems 1. Breast Cancer

a 4HPR as a Single Agent Oral 4HPR reduced the overall incidence of breast cancer and increased the latency to cancer development in carcinogentreated rats (Moon et al., 1979). Although it was not as potent as retinyl acetate, the toxicity was significantly less. Evaluation of the mammary gland confirmed the antiproliferative effects of 4HPR on mammary tissue. There was decreased ductal branching and end bud proliferation in 4HPR-treated rats compared to rats receiving no retinoid. The efficacy and safety data reported in this study prompted further studies of 4HPR as a chemopreventive agent. In later studies, 4HPR suppressed the development of subsequent mammary cancer following the removal of carcinogen-induced first palpable tumor. In a different mammary tumor system using mice, oral 4HPR administration suppressed genesis of spontaneous C3H mouse mammary tumors in nulliparous mice, although the difference between the control and the 4HPR

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group did not reach statistical significance. More importantly, 4HPR failed to inhibit mammary tumors in multiparous C3H mice. This discrepancy was explained by the presence of overt precancerous mammary lesions, referred to as hyperplastic alveolar nodules (HAN) in multiparous animals. It appears that 4HPR is ineffective in suppressing the progression of HAN to adenocarcinomas (Welsch et al., 1983). In accord with the above results, in vivo studies in virgin female C3H/He mice suggested antiproliferative activity of 4HPR since HAN development in mammary glands was reduced by 50% in animals maintained on 4HPR-supplemented diets compared to controls (Moon et al., 1983). These effects on mammary glands probably were independent of hormone levels since retinoid administration has no significant effect either on prolactin levels (Welsch et al., 1980) or ovarian function (Moon et al., 1979). Therapeutic efficacy of 4HPR was demonstrated in a rat study in which 4HPR resulted in complete regression of carcinogen-induced first mammary tumors in 22% of animals and partial regression in 19% of animals (Dowlatshahi et al., 1989). b. Combinations Containing 4HPR In an attempt to enhance the efficacy of 4HPR, various combinations have been investigated in animal systems. The combined effect of ovariectomy and 4HPR was compared to either treatment alone in a system utilizing N-methyl-N-nitrosourea (MNU) and 7,12-dimethylbenz[a]anthracene(DMBA) as chemical inducers of rat breast tumors (McCormick et al., 1982b). 4HPR was given to rats beginning at Day 7 postcarcinogen. Combined treatment was significantly more active in suppressing cancer induction than either treatment alone. 4HPR was a more effective inhibitor of carcinogenesis in ovariectomized animals than in intact animals, suggesting that 4HPR inhibition of carcinogenesis is not mediated via alteration in estrogens. 4HPR treatment also resulted in increased tumor latency and reduction in the number of cancers per rat. These data clearly demonstrated the chemopreventive ability of 4HPR in a rat model and its synergism with ovariectomy even though the exact mechanism of such synergy is unknown. In another study using DMBA as a carcinogen, rats were treated with oral calcium glucarate (CGT) and 4HPR alone or in combination, beginning 2 weeks prior to DMBA (Abou-Issa et al., 1988). Eighteen weeks later the incidence of tumor formation was observed. Both CGT and HPR, when administered alone at optimal doses, reduced the tumor incidence by 50 and 57%, respectively, and tumor multiplicity by 50 and 65 YO,respectively. When given in combination at suboptimal ineffective individual doses (onehalf the effective dose or less), synergism was seen, with 55-60% inhibition. The authors suggested that 4HPR may be the actual inhibitor of the rat mammary carcinogenesis, with CGT acting in an adjuvant role. The exact mechanism of synergism to inhibit the induction of the rat tumors by the

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HPR and CGT combination is not known. In a further extension of the study, the same CGT + HPR combination was evaluated in established rat mammary tumors induced by DMBA (Abou-Issa et al., 1989). Once the tumors reached 2 cm in size, the rats were given 4HPR and CGT alone or in combination. After 25 days of treatment, tumor growth was compared to control rats. CGT and 4HPR, when given alone in optimal doses, reduced the tumor sizes by 15 and 20%, respectively. In contrast, the combination of CGT and HPR acted synergistically to markedly inhibit the growth of mammary tumors (33% reduction compared to 98% increase in control rats). With the combination, reduction in tumor size was seen in 80% of the rats. The regimen was nontoxic and the estrogen-progesterone profile in control and combination-treatment groups did not show a significant difference. CGT4HPR represents an interesting combination since certain vegetables and fruits contain glucarate. A tamoxifen-HPR combination yielded interesting results in a study conducted by Ratko and associates (1989). MNU injection was used to induce mammary tumors in rats. The primary carcinoma was surgically removed and immediately following the removal, rats were started on a diet containing tamoxifen, 4HPR, a combination of both, or placebo. At Day 180, surviving rats were sacrificed. When compared to 4HPR or tamoxifen alone, treatment with the combination was more effective, as demonstrated by enhanced terminal survival and reduction in nonrecurrent cancer incidence and multiplicity. The incidence of first through fifth additional cancers was reduced by the combination treatment after surgical removal of primary carcinogen-induced tumor. When compared to single treatments, the combination was immediately and increasingly more effective in suppressing the appearance of additional lesions. In contrast to the effect of 4HPR or tamoxifen alone, which appears to be at the early stages of promotion or progression, the combination treatment appeared to be more efficacious a t all stages of promotion or progression. Not all combination studies with 4HPR and other agents have reported synergism. In one study, rats were given one of the following test diets containing (1) 4HPR alone, (2) selenium alone, (3) vitamin E + selenium, or (4) 4HPR + selenium vitamin E, 12 days prior to DMBA administration. At 140 days all treatment groups showed a decrease in tumor incidence, but none of the differences reached statistically significant levels. Tumor numbers were significantly reduced by 4HPR, selenium, and 4HPR + selenium + vitamin E, and tumor latency was extended in the selenium alone and 4HPR selenium + vitamin E groups. Combinations of different agents, therefore, failed to provide greater chemopreventive effect than individual agents alone (Cohen and Mahan, 1989). In a rat model using MNU as a mammary carcinogen, 4HPR and maleic anhydride-divinyl ether copolymer (MVE-2) were effective inhibitors of carcinogenesis when administered alone; however, no additive or synergistic

+

+

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activity was noted when both agents were administered as a combination (McCormick et al., 1982a). Monitoring serum 4HPR levels may explain the variable responses seen in the different 4HPR combinations described above. The lack of synergistic effect of a particular combination may not be related to their chemopreventive effects, but may be a result of altered metabolism or tissue availability of one or both agents (McCormick et al., 1982a). c. Summary of Breast Studies In animal models, 4HPR administration

resulted in both decreased carcinogen-induced tumor incidence and tumor multiplicity. 4HPR has also been shown to reduce tumor recurrence and delay tumor latency after removal of the first tumor. Additionally, 4HPR showed synergism with ovariectomy and tamoxifen in inhibition of carcinogen-induced tumors. 4HPR was active against established mammary tumors. These exciting data from animal studies have resulted in great enthusiasm to carry out similar chemopreventive studies in humans. 2. Bladder Cancer Becci and associates (1980) have reported inhibition of bladder carci(OH-BBN) in noma induction by N-butyl-N-(4-hydroxybutyl)nitrosamine 4HPR-treated mice. Animals received OH-BBN via gastric intubation. Highly invasive bladder carcinomas (similar to human transitional bladder carcinoma) were induced after treatment with OH-BBN twice a week for 9 weeks. Starting 1week after final carcinogen administration, animals were fed different diets. The 4HPR-containing diet significantly reduced cancer incidence. 4HPR was one of the most active and least toxic agents of 15 retinoids studied in another study of the OH-BBN transitional cell carcinoma model in mice (Moon et al., 1982). The all-trans and 13-cis isomers of 4HPR were found to be equally active, but the former compound was better tolerated than the latter. The combination of 4HPR with a maleic anhydride-divinyl ether copolymer (MVE-2) has been studied in mice using OH-BBN as a carcinogen. Both 4HPR and MVE-2 were found to be effective inhibitors of chemical carcinogenesis in this mouse model, but the chemopreventive efficacy of the combination was less than that of either 4HPR of MVE-2 alone (McCormick et al., 1982a). 3. Prostate Cancer

The effect of 4HPR on spontaneously developing prostate tumors was demonstrated in ACUsegHapBR rats. The incidence of prostate tumors was lower in 4HPR-treated rats compared to controls (27.5 vs 43.2%); however, the difference did not reach statistical significance (Oshirna et al., 1985). The ability of 4HPR to prevent prostate cancer was clearly demonstrated in the Lobund-Wistar rat model (Pollard et al., 1991). The animals were

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treated with oral 4HPR 7 months after initiation of methylnitrosourea and testosterone treatments. Among the control rats, 88% developed tumors compared to only 21% in the 4HPR-treated group. Thus, in this model, 4HPR caused marked inhibition of prostate carcinogenesis. The incidence of metastases was also significantly reduced. The authors suggested that the effect of 4HPR was possibly directed at the promotional stage of tumorigenic process since the 4HPR treatment was started 7 months after the initiation of carcinogen treatment. 4HPR may also inhibit tumor progression by inhibiting tumor growth through direct cytotoxic effects, as well as by inhibition of angiogenesis. In vivo, oral 4HPR was effective in inhibiting anaplastic rat prostate tumor growth a t an early stage, without any signs of toxicity (Pienta et al., 1993). In a mouse prostate reconstitution model system, 4HPR reduced YUS+ myc-induced prostate cancer incidence by 49% and tumor mass by 52% (Slawin et a/., 1993). In summary, 4HPR appears effective in inhibiting prostate carcinogenesis as well as early tumor growth in rat and mouse models. Thus, it is a promising agent to study in human prostate cancer prevention and early prostate cancer treatment. 4. Other Cancers

Skin cancer incidence was studied in a two-stage skin cancer model in CD-1 and SENCAR mice (McCormick and Moon, 1986). Mice received DMBA as an initiator and 12-0-tetradecanoylphorbol-13-acetate(TPA) as a promoter. Dietary 4HPR inhibited tumor progression and multiplicity, but, interestingly, the antipromotional activity of 4HPR was related to TPA dose. In a more important observation from the same study, 4HPR did not exhibit skin tumor-promoting activity which had been previously reported with the topical application of retinoic acid. In another report, oral 4HPR significantly reduced spontaneously occurring skin and subcutaneous tissue tumors in ACIIsegHapBR rats (Oshima et al., 1985). Carcinogen-induced pancreatic adenomas in female hamsters were reduced by a 4HPR-containing diet (Birt et al., 1981).4HPR demonstrated antilymphoma activity in the murine moloney lymphoma model (Dillehay et al., 1986). The incidence and number of carcinogen-induced colonic adenomas was reduced by oral 4HPR administration in a rat model (Silverman et al., 198 l). Interestingly, intrarectal administration of 4HPR demonstrated no such effect. Recently, 4HPR has been found to inhibit diethylnitrosamine (DEN)induced lung carcinogenesis in the hamster, but no significant effect of 4HPR was seen on MNU-induced tracheobronchial carcinogenesis in the same species (Moon, 1993). 4HPR is also effective against DEN-induced liver carcinogenesis in BALB/c mice; however, it does not inhibit esophageal turnorigenesis (Moon, 1993). In a human ovarian carcinoma xenograft

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model, intraperitoneal administration of 4HPR increased survival of the treated mice and enhanced the antitumor activity of cisplatin (Formelli and Cleris, 1993).

C. Timing and Duration of Retinoid Administration As the earliest stages of tumor development cannot be identified in humans, McCormick and Moon (1982) have done excellent studies in rats to determine efficacy of tumor inhibition utilizing retinoids administered at various time intervals after carcinogen exposure. MNU was used as a carcinogen and retinyl acetate was administered in the diet at 1,4, or 8 weeks after MNU injection. At high MNU doses retinyl acetate most effectively inhibited induction, as seen by increased tumor latency from 61 days to 116 days, and reduction in tumor number by 40% when treatment was begun 1week after MNU. Delaying retinyl acetate until 4 weeks resulted in slightly reduced chemoprevention efficacy (tumor latency, 88 days), but delay for 8 weeks resulted in loss of chemoprevention efficacy. The stage of precancerous lesion at the beginning of retinoid administration may be an important factor in determining the efficacy. The more advanced stage of preneoplasia may explain loss of efficacy in the 8-week group. Another study has been reported using an OH-BBN-induced bladder carcinoma model in which up to a 9-week delay in starting retinoid feeding did not diminish the ability of 13-cis-retinoic acid to inhibit bladder carcinogenesis (Becci et al., 1979). The duration of retinoid administration also appears to be of critical importance as is evident by the results of a study in rats by Thompson et al. (1979). Retinyl acetate, when administered continuously beyond 60 days, significantly prolonged cancer latency in retinoid responsive animals (those without palpable tumors at 60 days) as compared to those animals which were removed from the retinoid diet at 60 days. The latter group, once removed from the retinoid treatment, began to develop cancers at a rapid rate and the number of cancers per animal at the termination of the study (182 days) was similar to that of the animals treated with placebo throughout the study. This data suggested that continuous prolonged administration of retinoids may be necessary to maintain chemopreventive efficacy. Interestingly, in animals with palpable tumors at 60 days, continuation of retinoid administration had little effect on additional mammary cancers. These findings indicate significant heterogeneity among the animals in their response to retinoid treatment.

D. Mechanism of Action: Investigation in Animal Models Various studies have tried to determine the mechanism of the chemopreventive effect of retinoids and 4HPR. Mehta and Moon (1980) reported

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selective inhibition of mammary cell DNA synthesis in carcinogen-treated animals with retinyl acetate, while DNA synthesis in noncarcinogen fed animals was not affected. These data confirmed in uitro evidence of selective inhibition of DNA synthesis in preneoplastic and neoplastic mammary cells by retinyl acetate, without any significant effect on normal mammary epithelial DNA synthesis (Feldman and Foster, 1979). The addition of 4HPR or retinoic acid to organ cultures of mouse mammary glands inhibits prolactininduced increases in DNA synthesis (Mehta et al., 1983). The cell-cycle phase may also be important for retinoid action. In mouse epidermal cell cultures, retinyl acetate inhibited DNA synthesis only if it was present during or before the GI phase of the cell cycle (Yupsa et al., 1977). Retinoids in experimental studies have been shown to reduce DNA synthesis (Mehta and Moon, 1980), inhibit tumor-promoter-induced ornithine decarboxylase activity (Verma and Boutwell, 1977), inhibit growthfactor-induced cell transformation (Todaro et al., 1978), and reduce RNA polymerase activity of mammary cancer nuclei (Mehta et al., 1988a). Retinoids may act as chemopreventive agents by regulating cellular differentiation. Retinoic acid is known to regulate expression of the squamous differentiation markers including various keratins, transglutaminase, involucrin, and loricrin. The regulation appears to occur at the transcriptional level but other mechanisms are possible. A model has been proposed for the regulation of squamous cell differentiation based on the observation that it is a multistep process (Jetten etal., 1993).The induction of irreversible growth arrest appears to be necessary for epidermal keratinocytes to express squamous-cell-specific genes. Irreversible growth arrest may require induction of specific growth arrest genes (GAGs) which then induce irreversible growth arrest as well as the expression of differentiation markers (DIFs) and transcriptional factors. Retinoic acid probably does not suppress the GAGs, but more likely influences the expression or function of various DIFs. Several studies have provided evidence to support the suppression of keratin and transglutaminase and other squamous-cell-specific genes by retinoids at the transcription level via retinoic acid receptors. In addition to keratinocytes, fibroblasts, melanocytes, inflammatory cells, and many other cells are also targets for retinoids. Further understanding of the molecular mechanisms of retinoid action will provide better strategies for drug design (Jetten et al., 1993). 4HPR and RA may act through different receptors, since RA-resistant HL-60R and NB-306 cells are fully susceptible to inhibitory effects of 4HPR (Delia et al., 1993). In addition, 4HPR induces rapid apoptosis in these cells at a time when no differentiation is detectable, suggesting that the effects of 4HPR may be a result of its ability to promote apoptosis rather than differentiation. HL-60R carries a point mutation in the ligand-binding domain of RARa codon 411 and does not express RARP, RARy, and RXRa mRNA (Robertson et al., 1992a,b). The resistance to RA in the NB-306

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cell line is associated with altered expression of the pml/RAR fusion protein (Dermine et al., 1993). The activity of 4HPR against both these cell lines suggests a distinct mechanism of action. This possibility is supported by additional observations in which 4HPR fails to trans-activate RARa, RARP, RARy, and RXRa. Recently a leukemic cell line resistant to 4HPR but susceptible to RA has been reported; however, no details about its receptor status are available (Delia et al., 1993). 4HPR has been demonstrated to lower circulating insulin-like growth factor I (IGF-I) levels in early breast cancer patients. IGF-I is a potent mitogen for breast cancer cells and higher expression of IGF-I has been found in breast cancer cells than in normal epithelium. This led to speculation that the decline in plasma IGF-I may be one of the mechanisms through which 4HPR exerts an anticarcinogenic effect (Torrisi et al., 1993). Immunologic mechanisms may also contribute to the antineoplastic activity of 4HPR (Villa et a!., 1993). Natural killer (NK) activity was found to be 1.73 times higher in 4HPR-treated breast cancer patients compared to those receiving placebo. The increase in NK activity is not due to an increased number of NK cells, but appears to be related to increased functional activity of existing cells. In addition, the authors demonstrated that increased NK activity is not mediated by increased IL-2 production. In summary, the exact mechanism of action of 4HPR as a chemopreventive agent remains unknown.

VII. Human Trials 4HPR is an attractive chemopreventive agent to study in humans because of its significant activity in in uitro and in vivo preclinical studies with concomitant low toxicity. Chemoprevention trials require a prospective design to study high-risk or healthy individuals over an extended period of time. The end points are usually reduction of cancer incidence in a treatment group compared to a placebo control group or the study of intermediate biomarkers, if available. The chemopreventive agent must be safe for prolonged administration, and compliance should be high. In contrast, a chemotherapeutic trial is conducted in patients with a diagnosis of cancer, with response rate or mortality as the end point of the study. The tolerance for toxicity is higher than in chemopreventive trials. In addition, the duration of treatment in chemotherapeutic trials is usually shorter than in chemopreventive trials (Szarka et al., 1994). In both types of trials, the development of the new agent should go through phase I, 11, and 111 trials before being accepted as a standard.

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A. Chemotherapeutic Trials In a recently reported trial using 300-400 &day of 4HPR in 31 patients with advanced melanoma or advanced breast cancer, there were no complete or partial responses (Modiano et al., 1990a). Two patients achieved mixed responses and 8 patients had disease stabilization. Reversible mucocutaneous toxicity occurred in 52% of patients and reversible nyctalopia developed in three patients. One of them had a decreased B-wave amplitude on scotopic electroretinogram. In addition, serum cholesterol and triglyceride levels were elevated in 40 and 20% of the patients, respectively. These elevated levels returned to baseline 2-4 weeks after discontinuation of therapy or reduction in the dosage. Another trial was reported using tamoxifen at 20 mg/day alone or in combination with 100, 200, 300, or 400 mg/day of 4HPR in metastatic breast cancer. The patients on 4HPR had a 3-day “drug holiday” every 4 weeks to allow retinol levels to normalize in an attempt to prevent night blindness. 4HPR was administered at bedtime to 15 patients. The median duration of treatment was 6 months. Four patients experienced grade 1 or grade 2 anemia and 2 patients had grade 1 platelet toxicity. One patient on 4HPR 100 mg/day had elevated liver enzymes after 6 months of treatment (grade 1) and a second patient had grade 3 hepatic enzyme toxicity. Four patients had minor elevations in serum creatinine. There was a decrease in cholesterol levels which may have been secondary to tamoxifen. One CR and four improvements were seen. Lack of visual and mucocutaneous toxicity might have been due to the drug holiday given to the patients. Another possibility is that these patients were told not to take 4HPR with meals since food composition has been shown to increase the bioavailability of 4HPR. The authors concluded that this combination was safe for administration and the study is continuing with plans to escalate the dose up to 700 mg/day of 4HPR (Cobleigh et al., 1993). A third trial investigated the effect of 4HPR on prostate cancer in humans (Chodak et al., 1993). Three groups of patients eligible for this trial included 13 patients with clinically localized previously untreated prostate cancer (group I), 7 men with rising prostate-specific antigen (PSA) after radical prostatectomy (group 11), and 6 men with rising PSA after hormonal therapy (group 111). The pretreatment PSA ranged from 0.9 to 645 ng/ml. 4HPR was administered orally a t 300 mg/day for 25 days followed by a 3-day rest period. After 6 months of therapy, the 4HPR dose was reduced to 200 mg/day. The drug has been well tolerated, except for one patient who developed retinopathy. In group I, 11, and 111 patients, PSA declined by more than 10% in 5/13, 2/7, and 0/6 patients, respectively, suggesting some biological activity of 4HPR against early prostate cancer. Progressive disease was seen in 2,3, and 5 patients in groups I, 11, and 111, respectively.

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Currently, 18 patients are continuing treatment and further accrual is in progress. These three trials did not show any significant activity of 4HPR as a chemotherapeutic agent against already-established cancer but provided important safety data in humans.

B. Chemopreventive Trials 1. Breast Cancer a. Toxicity Trial 4HPR has been proposed for evaluation in humans as a chemopreventive agent. In order to select a dose that could be administered over a prolonged period of time, a randomized trial was performed utilizing 101 patients who underwent treatment for lymph-node-negative breast cancer 1-3 years prior to enrollment (Costa et al., 1989). At the time of study there was no evidence of relapse. During the first 6 months of the study, the patients were randomized to four groups to receive placebo, 100, 200, or 300 mg 4HPR daily. After 6 months, all patients received the same dose, 200 mg 4HPR per day, for up to 42 months orally with a 3-day drug interruption at the end of each month. One hundred patients were evaluable a t 6 months and 84 at 1 year. The toxicities encountered are listed in detail in the human toxicity section. Based on minimal side effects and good tolerance of prolonged administration, the authors selected 200 mg/day as the dose level for their long-term chemoprevention trial (Costa et al., 1989).

b. Efiicacy Trial Despite a wealth of data in preclinical studies, no human chemopreventive study using 4HPR has been completed and reported as of yet. In a patient treated for early breast cancer, the risk of developing contralateral cancer is about 0.8% per year for at least the next 10 years (Costa, 1993). Recently, a large randomized trial has been started in Milan using 4HPR as a chemopreventive agent in breast cancer. The aim of this study is to evaluate the effectiveness of 4HPR in preventing the incidence of contralateral primary breast cancer in patients with previously treated early breast cancer. Patients are randomized to one of two arms: a control arm, which receives no further treatment, or an intervention arm, which receives 4HPR at 200 mg/day with a 3-day drug holiday a t the end of each month for 5 years. An additional 2-year follow-up period is planned for both groups. In addition to physical examination, patients will be followed by yearly mammogram, biopsy of suspicious lesions, chest X-ray, and various blood tests including 4HPR, MPR, and retinol levels. Toxicity will be monitored along with abnormal laboratory values, and treatment will be interrupted for persistent mild side effects or moderate side effects. According to the information available, 2438 patients had been enrolled as of August 1991, and 1931 patients were being followed. In 17 patients, treatment has

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been discontinued for the following reasons: liver toxicity in 2, visual toxicity in 6, dermatosis in 4, allergies in 4, and enteritis in 1. Seven additional patients had possible toxicities requiring discontinuation of 4HPR. Four years after 4HPR administration, no changes in the mammographic patterns have been observed (Cassano et al., 1993). The initial group of patients is completing 5 years of treatment and will begin a 2-year observation period before the efficacy data become available (Costa, 1993). Because of the preclinical data demonstrating synergy between tamoxifen and 4HPR (Ratko et al., 1989) and a human trial addressing safety of such a combination (Cobleigh et al., 1993),a study to evaluate the combination of tamoxifen and 4HPR represents a logical next step in breast cancer chemoprevention. 2. Bladder Cancer

Several retinoids, including 4HPR and etretinate, have been utilized in bladder cancer chemoprevention trials. 4HPR is being investigated in bladder cancer chemoprevention because of superior tolerance. A pilot trial using 200 mg/day of 4HPR in 12 patients with previously resected superficial bladder cancer has been reported (Decensi et al., 1992). Seventeen patients served as controls and DNA content analyses by flow cytometry and conventional cytology were used as intermediate markers. At 12 months patients with DNA aneuploid stemlines decreased from 58 to 45% in the 4HPRtreated group, while increasing from 41 to 59% in the control group. In patients with stable DNA diploid profiles, the mean S phase and G, + M phase fractions declined with retinoid treatment. Three of 12 patients had suspicious cytology prior to 4HPR treatment which returned to normal after 4HPR administration. The control group showed an increase in patients with suspicious cytology from 24 to 35%. Four patients had impaired dark adaptation and one-third had transient dermatological alterations. The use of retinoids in bladder cancer prevention appears to be a promising field in which to do further studies. It is important to emphasize, however, that the relevance of DNA content as an intermediate marker has not been determined in human trials. 3. Head and Neck Cancer

Aerodigestive tumors appear to be susceptible targets for chemoprevention. Research using retinoids as chemopreventive agents in head and neck malignancies has shown promising results. A study was performed using 13-cis-retinoic acid as an adjuvant to primary therapy for head and neck cancer. Patients were randomized to a control arm or 50-100 mg/m2 of 13-cis-retinoic acid. There was no difference in recurrence rate between the two groups. However, a significant reduction was seen in the incidence of second primaries in patients taking the drug, 28% in controls versus 6% in the 13-cis-retinoic acid group (Hongetal., 1990).The benefit was achieved

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at the expense of significant dermatological toxicity (63%) and elevated triglyceride levels (27%). As 4HPR appears to be better tolerated than other retinoids, it is logical to use it in head and neck cancer prevention. Preliminary results of a randomized trial of 4HPR in patients with surgically treated oral leukoplakia, a premalignant lesion, have been reported (Chiesa et al., 1992). One hundred fifteen patients were randomized to 200 mg/day of 4HPR for 52 weeks versus no treatment after surgical resection of the lesion. Only 3 relapses or new lesions were seen in the 4HPR group compared to 12 in the control group at completion of 1 year. Only five patients interrupted treatment because of toxicity. The authors concluded that 4HPR is well tolerated and seems efficacious in preventing relapses and new localizations during the treatment period, but confirmation of these findings is required. 4. Skin Cancer

Several trials using different retinoids have not provided a clear answer regarding chemopreventive efficacy of retinoids in skin cancer (Kraemer et al., 1988, 1990; Tangrea et al., 1992; Greenberg et al., 1990). Ongoing trials using newer retinoids should provide important information in skin cancer chemoprevention. In Europe, a trial is evaluating 4HPR in patients with resected basal cell carcinoma of head and neck. 4HPR is given at 200 mg/day for 12 months and end points of the study are relapse of prior cancer and occurrence of new primary skin cancer. Four hundred and ten patients have already been randomized but results have not been published (Pastorino, 1992). 5. Prostate Cancer

We have undertaken a study in which 22 men deemed to be at high risk for the development of prostate cancer were given 100 mg/day of 4HPR for 1 year. No visual toxicity has occurred and only one patient has had to be taken off the study due to increased triglycerides. It appears that 4HPR is a safe agent at this low dose in this population of men ages 55-75 years and higher doses are being pursued (K. J. Pienta, unpublished data).

C. Human Toxicity In any chemoprevention, trial toxicity assumes significant importance since the drug is administered to healthy individuals. Vitamin A in large doses can cause major toxicities including liver toxicity, central nervous system abnormalities, bone abnormalities, and mucocutaneous problems. 4HPR appears to be less toxic than vitamin A. The following is a brief summary of side effects reported from various human studies.

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I. Ocular Toxicity

Ophthalmic toxicity remains a prime concern with nyctalopia, or impaired dark adaption, being the most common side effect. Retinograms have proven useful in detecting impaired dark adaptation prior to clinical symptoms in some patients. The first few patients to develop abnormal retinal function were receiving 800 mg/day of 4HPR for basal cell carcinoma (Kaiser-Kupfer et al., 1986). Two patients complained of visual symptoms after approximately 2 weeks of therapy, accompanied by changes in the electroretinogram. The discontinuation of 4HPR resulted in relief of symptoms. In both patients, the onset of symptoms, as well as their resolution, was rapid, suggesting a reversible mechanism. Three other patients had no visual symptoms. In another study using 300-400 mg/day of 4HPR, 3 of 3 1 patients developed reversible nyctalopia (Modiano et al., 1990a,b). Results of a larger study with a longer follow-up suggest that ocular toxicity is dose dependent with minimal incidence at the 200 mg/day dose level (Rotmensz et al., 1991). In the early phase of this study 4 of 101 patients had visual disturbances, 2 from the 300-mg group and 2 from the 200-mg group. One of these patients in the 300-mg group had marked visual difficulty at low levels of illumination after 6 months of treatment. Examination revealed corneal changes resembling those seen in patients taking amiodorone, accompanied by marked reduction of scotopic B-waves in both eyes on a dark-adapted electroretinogram. Two days after discontinuation of 4HPR, symptoms disappeared completely and the electroretinogram returned to almost normal after 9 days. None of the 11 other patients on whom retinograms were performed had altered dark adaptation. Visual impairment was not seen in the 200-mg group even after 1year of treatment. In the later phase of this study, the incidence rate of pathological retinogram findings was 6.1% at 37-42 months of 4HPR treatment and retinol levels were low in these subjects. A total of 5 3 patients underwent ophthalmic evaluation at 42 months. Seven patients reported impaired dark adaptation, but only 3 were confirmed by retinogram and all 3 had low retinol serum levels compared to the placebo group. The first two patients developed abnormalities at 24 and 40 months and both were able to resume 4HPR after a 4-month interval when the retinogram returned to normal. The third patient developed abnormal retinogram findings at 4 1 months, and 9 months after interruption of the treatment she still had some abnormality in one eye. On discontinuation of the drug, in most cases, these abnormalities were completely reversible. In an important study, 4HPR administration at 200 mg/day to stage I breast cancer patients resulted in alterations of dark adaptations in 50% of 4HPR-treated patients compared with 6 % of the controls (Decensi et al., 1994). However, half the patients with altered dark adaptation were asymp-

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tomatic. The alterations in dark adaptations inversely correlated with plasma retinol levels, with a threshold level of 10-16 pgldl. A complete normalization of dark adaptation resulted in all patients after 1month of drug interruption or vitamin A supplementation, but not after 3-day drug suspension. Conjunctival cytology alterations were seen in a higher number of patients taking 4HPR compared to controls (31% vs 13%), but no cases of keratoconjunctivitis were seen. The role of vitamin A supplementation in reducing the ocular toxicity of 4HPR needs to be confirmed in larger trials. Also, drug holidays may reduce the incidence of ocular problems. Retinograms should be done at the first suspicion of visual side effects since discontinuation of 4HPR appears to result in complete recovery. 2. Dermatological Toxicity

Skin toxicity is seen much less frequently with 4HPR than with other retinoids. Mild pruritus, mild alopecia, nail fragmentation, and xerosis have been reported. A widespread, painful morbilliform skin eruption and sporadic episodes of diarrhea have been reported in a patient with basal cell carcinoma being treated with 800 mg/day of 4HPR (Gross et al., 1991). Similar cutaneous eruptions have been reported with 4HPR treatment in psoriasis at 600 mg/day (Kingston et al., 1986). The Italian study at 200 mg/day of 4HPR did not report any similar cutaneous eruptions; however, dermatological complaints were the most common symptoms reported and included pruritus in 22 cases, skin dryness in 16 cases, and cheilitis and mouth dryness in 4 cases. In two patients receiving 200 mg, peeling of the palms and soles was evident after 5 months. Continuation of 4HPR up to 42 months resulted in minor dermatological problems including partial alopecia in six, nail fragmentation in five, xerosis in two, and urticaria and pruritus in one each (Rotmensz et al., 1991). 3. Psychological Effects

Pathological scores on anxiety and depression scales have been seen with 4HPR treatment, but no clinically evident psychological adverse reactions have been reported. In an Italian study, psychological evaluation was done in 40 patients, first at 4-5 months, and then at 36-42 months after 4HPR treatment initiation. Pathological scores were seen in 33 and 43.5% patients on an anxiety score and 40 and 47.5% patients on a depression scale, suggesting no significant difference between two evaluations. The difference found on the self-scoring mood questionnaire (15.5 a t baseline versus 6.5 at 42 months) was interpreted by authors as a decrease in enthusiasm for preventive treatment with the passage of time (Rotmensz et a!., 1991).

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4. Hepatic Toxicity Seven of 101 patients treated with 4HPR had increases in liver enzymes 2 to 4 times above baseline, but no serious liver toxicity has been seen so far. In one patient with elevated transaminases, liver biopsy showed diffuse steatosis, but markers were also positive for hepatitis B (Costa et al., 1989). I. Miscellaneous

An increase in triglyceride and cholesterol levels has been reported following 4HPR treatment in breast cancer patients (Modiano et al., 1990a). In contrast to this study, the chemopreventive study did not show any significant differences in cholesterol, triglyceride, LDL, or HDL levels between control and treatment groups after 8 months (Pizzichetta et al., 1992). A mild increase in triglyceride levels was seen in 4 of 79 patients but did not require dose alteration (Rotmensz et al., 1991). A bone-density evaluation was done in 4 7 patients at 42 months of 4HPR treatment in the chemopreventive study. Six had pathological and another 6 had borderline values, but no fractures were seen. Five of these patients had additional risk factors for bone-density loss. From the available information no definite conclusions can be drawn about changes in bone density (Rotmensz et al., 1991). From the same study, dyspepsia, muscle pain, headache, dizziness, and pruritus were reported as rare. Necrotizing vasculitis, which was reported in the literature, has not been seen in chemoprevention trials (Costa, 1993). The spectrum of congenital defects induced by synthetic retinoids are similar to those induced by retinoic acid (Flanagan et al., 1987); however, more data are needed to study the effect of the 4HPR on carcinogenicity and teratogenicity in humans. Summarizing available data on 4HPR, it appears that 4HPR can be safely administered at 200 mg/day for prolonged periods without significant toxicity; however, at the first evidence of visual problems, drug interruption or even discontinuation may be necessary for individual patients.

VIII. Future Directions The role of 4HPR as a chemopreventive agent is evolving. There is increasing evidence that retinoids will have an important role in cancer chemoprevention. The results of the Milan breast cancer prevention study are eagerly awaited. The combination of tamoxifen and 4HPR is a next logical step in breast cancer prevention, since in vivo animal data are promising. 4HPR appears promising in bladder cancer chemoprevention but more information is needed in human trials. 4HPR is being studied in human trials in head and neck cancer, where tremendous opportunities exist to prevent second cancers. In the future, combinations of two or more chemopreventive agents with different mechanisms of action may provide an oppor-

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tunity to block carcinogenesis at multiple steps and, thus, may provide more effective cancer prevention.

IX. Summary It has been suggested that ultimately half of all cancers might be prevented by early interventions (Costa, 1993). 4HPR has been shown to be an effective and safe agent in various in vivo animal trials and well tolerated in human trials. At present multiple clinical trials are assessing its efficacy in preventing a variety of cancers. References Abou-lssa, H. M., Duruibe, V. A., Minton, J. P., Larroya, S., Dwivedi, D., and Webb, T. E. (1988). Putative metabolite derived from dietary combinations of calcium glucarate and N(4-hydroxyphenyl) retinamide act synergistically to inhibit the induction of rat mammary tumors by 7,12 dimethylbenz[a]anthracene. Proc. Nut/. Acad. Sci. USA 85,4181-4184. Abou-lssa, H., Webb, T. E., Minton, J. P., and Moeschberger, M. (1989). Chemotherapeutic evaluation of glucarate and N-(4-hydroxyphenyl)retinamidealone and in combination in the rat mammary tumor model. /. Nut/. Cancer Inst. 81, 1820-1823. Batres, R. O., and Olson, J. A. (1987). A marginal vitamin status alters the distribution of vitamin A among parenchymal and stellate cells in rat liver. /. Nutr. 117, 874-879. Becci, P. J., Thompson, H. J., Grubbs, C. J., Brown, C. C., and Moon, R. C. (1979). Effect of delay in administration of 13-cis-retinoic acid on the inhibition of urinary bladder carcinogenesis in the rat. Cancer Res. 39, 3141-3144. Becci, P. J., Thompson, J. H., Sporn, M. B., and Moon, R. C. (1980). Retinoid inhibition of highly invasive urinary bladder carcinomas induced in mice by N-butyl-(n-4hydroxybuty1)nitrosamine (OH-BBN). Proc. Am. Assoc. Cancer Res. 21, 88. Bertram, J. S., Mordan, L. J., Blair, S. J., and Hui, S. (1981). Effects of retinoids on neoplastic transformation, cell adhesion and membrane topography of cultured 10T1/2 cells. Ann. N. Y. Acad. Sci. 359, 218-236. Birt, D. F., Sayed, S., Davies, M. H., and Pour, P. (1981). Sex difference in the effects of retinoids on carcinogenesis by N-nitroso-2-oxypropyI)amine in Syrian hamsters. Cancer Lett. 14, 13-21. Blaner, W. S . (1993). Biochemistry and pharmacology of retinoids. In “Retinoids in Oncology” (W. K. Hong and R. Lotan, eds.), pp. 1-42. Marcel Dekker, Inc., New York. Bollag, W., and Matter, A. (1981). From vitamin A to retinoids in experimental and clinical oncology: Achievements, failures and outlook. Ann. N. Y. Acad. Sci. 359, 9-23. Bollag, W., and Peck, R. (1993). Modulation of growth and differentiation by combined retinoid and cytokines in cancer. In “Retinoids In Oncology” (W. K. Hong and R. Lotan, eds.), pp. 89-108. Dekker, Inc., New York. Boone, C. W., Kelloff, G. J., and Malone, W. F. (1990). Identification of candidate cancer chemopreventive agents and their evaluation in animals models and human clinical trials. Cancer Res. 50, 2-9. Cassano, E., Coopmans de Yoldi, G., Ferranti, C., Costa, A., Mascotti, G., De Palo, G., and Veronesi, U. (1993). Mammographic patterns in breast cancer chemoprevention with fenretinide (4-HPR). Eur. /. Cancer 29A, 2161-2163. Chiesa, F., Tradati, N., Marazza, M., Rossi, N., Boracchi, P., Mariani, L., Clerici, M., Formelli,

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