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The antitumor effects of N-(4-hydroxyphenyl)retinamide (4-HPR), and its stable C-linked analog, 4-hydroxybenzylretinone (4-HBR) on the regression of established 7,12-dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors were compared. 4-HBR is a stable and nonhydroyzable derivative which cannot be converted in vivo to retinoic acid (RA). The results indicate that 4-HBR decreased mammary tumor volumes to the same extent as equimolar concentration (2 mmol/kg diet) of 4-HPR (-45% for 4-HBR vs. -42% for 4-HPR, p<0.01). Both 4-HPR and 4-HBR bind very poorly to nuclear retinoid receptors RARs and RXRs. The similarity of physicochemical properties of 4-HPR and 4-HBR as well as their equal antitumor potency suggests that 4-HPR like 4-HBR, is acting directly rather than through hydrolysis to free RA. Treatment with 4-HPR caused an almost 65% decrease in serum retinol levels. These results suggest that 4-HBR may have a significant chemotherapeutic advantage over 4-HPR, as the nonhydrolyzable analog may not cause night blindness which occurs as a significant side effect of 4-HPR usage.

The long term chemopreventive effects of the N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG), and its stable C-linked benzyl glucuronide analog, retinamidobenzyl glucuronide (4-HPRCG) on the growth and development of 7,12-dimethylbenz[a]anthracene-induced mammary tumors were compared. The retinamidobenzyl glucuronide is stable toward acid hydrolysis and resists the actions of beta-glucuronidase. The results indicate that the C-linked glucuronide analog, 4-HPRCG has a greater chemopreventive potency than an equimolar concentration of 4-HPROG. Tumor latency was 15% longer in rats fed 2 mmol/kg diet of 4-HPRCG as compared to 4-HPROG. At 80 days post DMBA-intubation, tumor incidence was 57% and 27% in the 4-HPROG and 4-HPRCG treated rats, respectively. Tumor multiplicity was also markedly decreased in the 4-HPRCG treated rats. At 80 days post DMBA intubation the control rats had an average of 1.43 tumors/rat compared to 0.71 and 0.36 tumors/rat in the 4-HPROG and 4-HPRCG respectively. The higher potency and low toxicity of 4-HPRCG suggest that this stable analog may have an in vivo chemopreventive advantage over its analog, 4-HPROG. The results also demonstrated that these glucuronide analogs do not bind effectively in vitro either to the nuclear retinoid receptors or to the cellular retinoid binding proteins. Regardless of the mode of action of these retinoids, they are clearly effective chemopreventive agents.

The growth inhibitory effects of N-(4- Hydroxyphenyl) retinamide (4-HPR) and its glucuronide derivative, N-(4-Hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) on established DMBA induced rat mammary tumors were compared. The results indicate that the glucuronide analog had a greater antitumor potency than equimolar concentration of the free retinoid. Tumor regression occurred in 75% of the rats fed 2 mmol/Kg diet of 4-HPROG. In a 6-week study, the maximum tolerated dietary dose (MTD) was found to be 3.5 mmol/Kg diet for 4-HPR and 5 mmol/Kg diet in the case of 4-HPROG. The higher potency and lower toxicity of the glucuronide suggests that this conjugate may have an in vivo chemotherapeutic advantage over the parent free retinoid.

The O-glucuronide analog of N-(4-hydroxyphenyl)retinamide (4-HPROG) has shown a greater chemopreventive activity than the parent N-(4-hydroxyphenyl)retinamide (4-HPR). However, this compound is relatively unstable. In order to improve stability and efficacy, we have prepared a number of stable C-linked analogs of 4-HPROG (C-phenyl and C-benzyl glucuronosyl, glucosyl, and xylosyl analogs). These analogs are stable toward acid hydrolysis and the glucuronosyl analogs resist the actions of beta-glucuronidase. The analogs were prescreened for their antiproliferative potential in vitro using cultured human MCF-7 breast cancer cells. Selected analogs were then evaluated for their ability to inhibit the development and growth of tumors in the 7,12-dimethylbenzanthracene-induced rat mammary tumor model. Although the stable C-linked analogs bound poorly to the nuclear retinoic acid receptors, many showed more potency than the less stable 4-HPROG in inhibiting tumor incidence and multiplicity in vivo. The glucuronide/glucoside analogs are more potent than the xylosides, and the C-benzyl more effective than the C-phenyl analogs. The higher potency of at least two C-linked analogs (retinamidobenzyl glucuronide and retinamidobenzyl glucose) suggests that these analogs may have a chemopreventive advantage over the parent retinamide and its natural O-glucuronide.

The independent effects of the potential cancer chemopreventive agent calcium glucarate (CGT) when fed (128 mmol/kg diet) during the initiation (I), promotion (P) or (I+P) phases of 7,12-dimethylbenzanthracene-induced rat mammary carcinogenesis, was compared to that of the known chemopreventive agent N-(4-hydroxyphenyl) retinamide (4-HPR) fed (2.0 mmol/kg diet) during these same phases. CGT and especially 4-HPR both significantly increased tumor latency when fed during the P-phase. When fed during I, P or I+P phases mammary tumor incidence was reduced compared to the controls 33%, 42% and 67% by 4-HPR and 18%, 42% and 50% by CGT. Similarly, tumor multiplicity was significantly reduced by either agent. For example, as compared to the corresponding control, when fed during the I, P or I+P phases 4-HPR reduced tumor multiplicity 63, 34 and 63%, while CGT reduced tumor multiplicity 28, 42 and 63% respectively. CGT, like 4-HPR, acts on both the I and P phases with the effect being maximal when fed during P and I+P phases.

In synergistic combination 0.75 mmol/kg diet of N-(4-hydroxyphenyl) retinamide and 32 mmol/kg diet of glucarate inhibits the growth of primary rat mammary tumors, but are equally effective as single agents at 1.5 and 128 mmol/kg diet, respectively. Dose-response studies suggest that like retinoids, glucarate acts directly on tumor cells, rather than having an adjuvant effect. Although synergism is maintained down to at least 0.38 mmol/kg diet of the retinoid, experiments using Vitamin A-deficient diets indicates 128 mmol/kg glucarate acts independent of retinoid. Both alone and in combination, glucarate and retinoid inhibited the growth of human mammary tumor cells grown in the athymic mouse, the growth of rat mammary tumors in germfree rats and the hormone-independent MTW 9a/R rat mammary tumor. Like retinoids, glucarate suppresses protein kinase C and induces transforming growth factor-beta, in the mammary tumor cells.

The inhibitory effects of N-(4-hydroxyphenyl) retinamide and its glucuronide derivative on the development and growth of 7,12-dimethylbenz (a) anthracene - induced rat mammary tumors in vivo were compared. The results indicate that the glucuronide had a greater chemopreventive potency than equimolar concentration of the free retinoid by all the criteria measured, mainly the inhibition of tumor incidence, multiplicity and tumor growth. HPLC analysis of the blood of the rats showed no hydrolysis of the glucuronide during its chronic consumption, indicating that the retinoid glucuronide is probably acting in vivo per se rather than through hydrolysis to the free retinoid. The higher potency and lower toxicity of the glucuronide suggests, for the first time, that the conjugate may have an in vivo chemopreventive advantage over the parent retinamide.

The biochemical basis for the cancer chemopreventive and anti-cancer activities of glucarate, retinoids (13-cis-retinoic acid, hydroxyphenyl retinamide) and their synergistic combination, has been evaluated. Neither alone nor in combination did these agents affect the level in the rat, of enzymes which are (a) known to correlate with reduced risk of carcinogenesis (detoxification enzyme, catalase, glutathione reductase) nor (b) enzymes which correlate with increased risk of carcinogenesis (beta-glucuronidase, xanthine oxidase, glucose-6-phosphate dehydrogenase). Retinoids, but neither glucarate nor its lactone inhibited free radical-induced lipid peroxidation. Both agents alone and synergistically in combination, raise cellular cAMP levels, repress protein kinase C and more generally inhibited DNA synthesis.

The Nb rat prostate adenocarcinomas - androgen insensitive II and III - were utilized in three experiments utilizing protamine sulfate, persantine (dipyridamole), and heparin-cortisone combination as agents evaluated for their ability to affect the incidence of systemic metastasis in this animal model. The results indicate that all agents tested were effective in reducing metastasis; protamine and persantine (10 mg/kg/day) being the most effective with 0% metastasis. In addition, animals treated with protamine had a statistically significant decrease in tumor volume. The mechanism responsible for this decrease remains unclear at present.