Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction.

BACKGROUND: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events. METHODS: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice. RESULTS: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions. CONCLUSIONS: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

TransCon IL-2 ß/γ: a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2 variant with improved pharmacokinetics and potent activation of cytotoxic immune cells for the treatment of cancer.

BACKGROUND: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe toxicities including cytokine storm and vascular leak syndrome (VLS). IL-2 promotes antitumor function of IL-2Rß/γ+ natural killer (NK) cells and CD8+, CD4+ and gamma delta (γδ) T cells. However, IL-2 also potently activates immunosuppressive IL-2Rα+ regulatory T cells (Tregs) and IL-2Rα+ eosinophils and endothelial cells, which may promote VLS. Aldesleukin is rapidly cleared requiring frequent dosing, resulting in high Cmax likely potentiating toxicity. Thus, IL-2 cancer immunotherapy has two critical drawbacks: potent activation of undesired IL-2Rα+ cells and suboptimal pharmacokinetics with high Cmax and short half-life. METHODS: TransCon IL-2 ß/γ was designed to optimally address these drawbacks. To abolish IL-2Rα binding yet retain strong IL-2Rß/γ activity, IL-2 ß/γ was created by permanently attaching a small methoxy polyethylene glycol (mPEG) moiety in the IL-2Rα binding site. To improve pharmacokinetics, IL-2 ß/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon (transient conjugation) linker creating a prodrug, TransCon IL-2 ß/γ, with sustained release of IL-2 ß/γ. IL-2 ß/γ was characterized in binding and primary cell assays while TransCon IL-2 ß/γ was studied in tumor-bearing mice and cynomolgus monkeys. RESULTS: IL-2 ß/γ demonstrated selective and potent human IL-2Rß/γ binding and activation without IL-2Rα interactions. TransCon IL-2 ß/γ showed slow-release pharmacokinetics with a low Cmax and a long (>30 hours) effective half-life for IL-2 ß/γ in monkeys. In mouse tumor models, TransCon IL-2 ß/γ promoted CD8+ T cell and NK cell activation and antitumor activity. In monkeys, TransCon IL-2 ß/γ induced robust activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells, Tregs and eosinophils, with no evidence of cytokine storm or VLS. Similarly, IL-2 ß/γ enhanced proliferation and cytotoxicity of primary human CD8+ T cells, NK cells and γδ T cells. SUMMARY: TransCon IL-2 ß/γ is a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2. It has remarkable and durable pharmacodynamic effects in monkeys and potential for improved clinical efficacy and tolerability compared with aldesleukin. TransCon IL-2 ß/γ is currently being evaluated in a Phase 1/2 clinical trial (NCT05081609).

Phase 1 safety, tolerability, pharmacokinetics and pharmacodynamics results of a long-acting C-type natriuretic peptide prodrug, TransCon CNP.

AIM: TransCon CNP is a novel prodrug designed to provide sustained release of C-type natriuretic peptide (CNP) for once-weekly therapy, addressing the pathology leading to aberrant skeletal development in achondroplasia. This phase 1 trial was initiated to assess the safety, tolerability, pharmacodynamics (PD) and pharmacokinetics (PK) of TransCon CNP. METHODS: This randomized, placebo-controlled, single-ascending dose phase 1 trial was performed at two sites in Australia and enrolled 45 healthy adult males. Subjects received placebo or TransCon CNP (single-ascending dose cohorts [3, 10, 25, 75 or 150 µg CNP/kg]). The primary endpoint was frequency of adverse events and other safety outcomes. Other endpoints included PK and PD measured by cyclic guanosine-monophosphate (cGMP) and amino-terminal propeptide of CNP (NTproCNP). RESULTS: TransCon CNP provided continuous systemic exposure to CNP over at least 7 days post-dose. Plasma and urine levels of cGMP were significantly increased in subjects administered TransCon CNP at 75-150 µg CNP/kg, indicating target engagement of active CNP at the natriuretic peptide receptor-B (NPR-B) for at least 1 week post-dose. TransCon CNP was well-tolerated, with no serious treatment-emergent adverse events or discontinuations. Extensive cardiac safety assessments did not reveal any clinically relevant effects on electrocardiogram parameters, including heart rate, PR, QRS and QTcF intervals. CONCLUSIONS: Safety and PD data from this phase 1 trial support that TransCon CNP is well tolerated, with a PK profile compatible with a once-weekly dosing regimen. Further studies are ongoing to evaluate the potential of TransCon CNP to positively impact abnormal endochondral ossification in children with achondroplasia.

Weekly Lonapegsomatropin in Treatment-Naïve Children With Growth Hormone Deficiency: The Phase 3 heiGHt Trial.

CONTEXT: For children with growth hormone deficiency (GHD), treatment burden with daily somatropin injections [human growth hormone (hGH)] is high, which may lead to poor adherence and suboptimal overall treatment outcomes. Lonapegsomatropin (TransCon hGH) is an investigational long-acting, once-weekly prodrug for the treatment of GHD. OBJECTIVE: The objective of this study was to evaluate the efficacy and safety of once-weekly lonapegsomatropin vs daily somatropin. DESIGN: The heiGHt trial was a randomized, open-label, active-controlled, 52-week Phase 3 trial (NCT02781727). SETTING: This trial took place at 73 sites across 15 countries. PATIENTS: This trial enrolled and dosed 161 treatment-naïve, prepubertal patients with GHD. INTERVENTIONS: Patients were randomized 2:1 to receive lonapegsomatropin 0.24 mg hGH/kg/week or an equivalent weekly dose of somatropin delivered daily. MAIN OUTCOME MEASURE: The primary end point was annualized height velocity (AHV) at week 52. Secondary efficacy end points included change from baseline in height SD scores (SDS). RESULTS: Least squares (LS) mean (SE) AHV at 52 weeks was 11.2 (0.2) cm/year for lonapegsomatropin vs 10.3 (0.3) cm/year for daily somatropin (P = 0.009), with lonapegsomatropin demonstrating both noninferiority and superiority over daily somatropin. LS mean (SE) height SDS increased from baseline to week 52 by 1.10 (0.04) vs 0.96 (0.05) in the weekly lonapegsomatropin vs daily somatropin groups (P = 0.01). Bone age/chronological age ratio, adverse events, tolerability, and immunogenicity were similar between groups. CONCLUSIONS: The trial met its primary objective of noninferiority in AHV and further showed superiority of lonapegsomatropin compared to daily somatropin, with similar safety, in treatment-naïve children with GHD.

Identification and preclinical development of an anti-proteolytic uPA antibody for rheumatoid arthritis.

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg-1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg-1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.

Design and Preclinical Development of TransCon PTH, an Investigational Sustained-Release PTH Replacement Therapy for Hypoparathyroidism.

Hypoparathyroidism (HP) is a condition of parathyroid hormone (PTH) deficiency leading to abnormal calcium and phosphate metabolism. The mainstay of therapy consists of vitamin D and calcium supplements, as well as adjunct Natpara (PTH(1-84)). However, neither therapy optimally controls urinary calcium (uCa) or significantly reduces the incidence of hypercalcemia and hypocalcemia. TransCon PTH, a sustained-release prodrug of PTH(1-34) in development for the treatment of HP, was designed to overcome these limitations. To determine the pharmacokinetics and pharmacodynamics of TransCon PTH, single and repeat s.c. dose studies were performed in rats and monkeys. TransCon PTH demonstrated a half-life of 28 and 34 hours in rats and monkeys, respectively. After repeated dosing, an infusion-like profile of the released PTH, characterized by low peak-to-trough levels, was obtained in both species. In intact rats and monkeys, daily subcutaneous administration of TransCon PTH was associated with increases in serum calcium (sCa) levels and decreases in serum phosphate levels (sP). In monkeys, at a single dose of TransCon PTH that increased sCa levels within the normal range, a concurrent decrease in uCa excretion was observed. In 4-week repeat-dose studies in intact rats and monkeys, uCa excretion was comparable to controls across all dose levels despite increases in sCa levels. Further, in a rat model of HP, TransCon PTH normalized sCa and sP levels 24 hours per day. This was in contrast to only transient trends toward normalization of sCa and sP levels with an up to 6-fold higher molar dose of PTH(1-84). After repeated dosing to HP rats, uCa excretion transiently increased, corresponding to increases in sCa above normal range, but at the end of the treatment period, uCa excretion was generally comparable to sham controls. TransCon PTH was well tolerated and the observed pharmacokinetics and pharmacodynamics were in line with the expected action of physiological replacement of PTH. © 2019 American Society for Bone and Mineral Research.

TransCon CNP, a Sustained-Release C-Type Natriuretic Peptide Prodrug, a Potentially Safe and Efficacious New Therapeutic Modality for the Treatment of Comorbidities Associated with Fibroblast Growth Factor Receptor 3-Related Skeletal Dysplasias.

TransCon CNP is a C-type natriuretic peptide (CNP-38) conjugated via a cleavable linker to a polyethylene glycol carrier molecule, designed to provide sustained systemic CNP levels upon weekly subcutaneous administration. TransCon CNP is in clinical development for the treatment of comorbidities associated with achondroplasia. In both mice and cynomolgus monkeys, sustained exposure to CNP via TransCon CNP was more efficacious in stimulating bone growth than intermittent CNP exposure. TransCon CNP was well tolerated with no adverse cardiovascular effects observed at exposure levels exceeding the expected clinical therapeutic exposure. At equivalent dose levels, reductions in blood pressure and/or an increase in heart rate were seen following single subcutaneous injections of the unconjugated CNP-38 molecule or a daily CNP-39 molecule (same amino acid sequence as Vosoritide, USAN:INN). The half-life of the daily CNP-39 molecule in cynomolgus monkey was estimated to be 20 minutes, compared with 90 hours for CNP-38, released from TransCon CNP. C max for the CNP-39 molecule (20 µg/kg) was approximately 100-fold higher, compared with the peak CNP level associated with administration of 100 µg/kg CNP as TransCon CNP. Furthermore, CNP exposure for the daily CNP-39 molecule was only evident for up to 2 hours postdose (lower limit of quantification 37 pmol/l), whereas TransCon CNP gave rise to systemic exposure to CNP-38 for at least 7 days postdose. The prolonged CNP exposure and associated hemodynamically safe peak serum concentrations associated with TransCon CNP administration are suggested to improve efficacy, compared with short-lived CNP molecules, due to better therapeutic drug coverage and decreased risk of hypotension. SIGNIFICANCE STATEMENT: The hormone C-type natriuretic peptide (CNP) is in clinical development for the treatment of comorbidities associated with achondroplasia, the most common form of human dwarfism. The TransCon Technology was used to design TransCon CNP, a prodrug that slowly releases active CNP in the body over several days. Preclinical data show great promise for TransCon CNP to be an effective and well-tolerated drug that provides sustained levels of CNP in a convenient once-weekly dose, while avoiding high systemic CNP bolus concentrations that can induce cardiovascular side effects.

Estrogen and phytoestrogens: Effect on eNOS expression and in vitro vasodilation in cerebral arteries in ovariectomized Watanabe heritable hyperlipidemic rabbits.

OBJECTIVES: To evaluate the effect of estrogen replacement therapy or soy isoflavones supplement on endothelium-dependent relaxation in vitro and gene expression of endothelial nitric oxide synthase (eNOS) in cerebral arteries in a rabbit model of human hypercholesterolemia. STUDY DESIGN: Thirty-six female ovariectomized Watanabe heritable hyperlipidemic (WHHL) rabbits were randomised to treatment with 17beta-estradiol (17beta-E(2)), SoyLife 150 or control for 16 weeks. Ring segments of basilar artery (BA) and posterior cerebral artery (PCA) were mounted in myographs for isometric tension recordings. Concentration response curves to carbamylcholine chloride, sodium nitroprusside (SNP) and l-NAME were evaluated after precontraction with potassium. Total RNA was extracted, reverse transcribed and eNOS quantitated by real-time polymerase chain reaction (real-time PCR). RESULTS: Plasma cholesterol was significantly higher at termination in the SoyLife group (P<0.0001), whereas low-density lipoprotein (LDL) cholesterol was comparable in all treatment groups. Neither treatment influenced the endothelium-dependent responses to carbamylcholine chloride or l-NAME or the endothelium-independent response to SNP in any of the arteries. Correspondingly, eNOS mRNA was similarly expressed in all treatment groups in both arteries. CONCLUSIONS: Improvement of cerebral endothelial function by estrogen or soy isoflavones in ovariectomized WHHL rabbits is not supported by the present data. The findings may be unique to the WHHL rabbit in which the hypocholesterolemic effect of estrogens mediated by upregulation of liver LDL receptors is excluded.

Estrogenic effect of soy isoflavones on mammary gland morphogenesis and gene expression profile.

We examined the effect of 17beta-estradiol (E2) and soy isoflavones' exposure on morphogenesis and global gene expression in the murine mammary gland. Three exposure regimens were applied: isoflavones added to the diet throughout either the lactational period (via the dams) or the postweaning period and E2 administered orally during the lactational period. Whole mounts of mammary glands were evaluated both in juvenile and adult animals with respect to branching morphogenesis and terminal end bud (TEB) formation. At postnatal day (PND) 28, we observed a significant increase in branching morphogenesis in all treated groups with the most pronounced effect after E2 exposure. For the E2-treated animals there was also a significant increase in TEB formation. At PNDs 42-43 the postweaning isoflavone and the E2 groups showed a transient reduction in the number of TEBs. A similar response after isoflavone and E2 exposure was further substantiated by changes in gene expression, since the same groups of genes were up- and downregulated, particularly in the E2 and postweaning isoflavone regimen. All changes in gene expression correlated with changes in the cellular composition of the gland, i.e., more and larger TEBs and ducts. The results suggest an estrogenic response of physiological doses of isoflavones on mammary gland development at both the morphological and molecular level, which resembled that induced by puberty.

Influence of Prevastein, an isoflavone-rich soy product, on mammary gland development and tumorigenesis in Tg.NK (MMTV/c-neu) mice.

We investigated spontaneous mammary tumor development and mammary gland morphogenesis in female Tg.NK mice postnatally exposed to dietary soy isoflavones (0, 11, 39, and 130 mg aglycones/kg diet) added to a Western-style diet. Instead of preventing mammary tumorigenesis, the highest dose of isoflavones was associated with a small but significant increase in the number and size of tumors as compared to mice administered a Western-style control diet (P < 0.05). At postnatal Week 6, dynamic activity (measured as apoptotic density) at the highest dose and the degree of branching of the mammary tree in all isoflavone-exposed groups was increased as compared to controls (P < 0.05). At adulthood, the epithelium appeared more quiescent in the medium- and high-dose groups evident by reduced apoptotic density and a reduction in the percentage of terminal end buds (TEBs), respectively, as compared to controls (P < 0.05). The number of actively dividing cells within the TEBs was unaffected by isoflavone exposure as was the activity of drug-metabolizing and antioxidant enzymes. In conclusion, isoflavones may augment mammary gland and mammary tumor development.

Anthocyanins increase low-density lipoprotein and plasma cholesterol and do not reduce atherosclerosis in Watanabe Heritable Hyperlipidemic rabbits.

Anthocyanin-rich beverages have shown beneficial effects on coronary heart disease in epidemiological and intervention studies. In the present study, we investigated the effect of black currant anthocyanins on atherosclerosis. Watanabe Heritable Hyperlipidemic rabbits (n = 61) were fed either a purified anthocyanin fraction from black currants, a black currant juice, probucol or control diet for 16 weeks. Purified anthocyanins significantly increased plasma cholesterol and low-density lipoprotein (LDL) cholesterol. Intake of black currant juice had no effect on total plasma cholesterol, but lowered very-low-density lipoprotein (VLDL) cholesterol significantly. There were no significant effects of either purified anthocyanins or black currant juice on aortic cholesterol or development of atherosclerosis after 16 weeks. Probucol had no effect on plasma cholesterol but significantly lowered VLDL-cholesterol and decreased aortic cholesterol accumulation. The erythrocyte antioxidant enzyme glutathione peroxidase was significantly increased by purified anthocyanins and superoxide dismutase was increased by both anthocyanin-containing treatments. Other markers of plasma antioxidant capacity, antioxidant enzymes, protein and lipid oxidation were not affected by any of the anthocyanin treatments. Adverse effects of purified anthocyanins were observed on plasma- and LDL-cholesterol. These effects were not observed with black currant juice, suggesting that black currants may contain components reducing the adverse effects of anthocyanins.

Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring.

Prochloraz is a commonly used fungicide that has shown multiple mechanisms of action in vitro. It antagonizes the androgen and the estrogen receptors, agonizes the Ah receptor, and inhibits aromatase activity. In vivo prochloraz acts antiandrogenically in the Hershberger assay by reducing weights of reproductive organs, affecting androgen-regulated gene expressions, and increasing luteinizing hormone (LH) levels. The purpose of this study was to investigate reproductive toxic effects after exposure during gestation and lactation to prochloraz alone and a mixture of five pesticides (deltamethrin, methiocarb, prochloraz, simazine, and tribenuron-methyl). Prochloraz (30 mg/kg/day) or the mixture (20 mg/kg/day) was dosed to pregnant Wistar dams from gestational day (GD) 7 until postnatal day (PND) 16. Some dams were taken for cesarean section at GD 21, and others were allowed to give birth. Results showed that prochloraz and the mixture significantly reduced plasma and testicular testosterone levels in GD 21 male fetuses, whereas testicular progesterone was increased. Gestational length was increased by prochloraz. Chemical analysis of the rat breast milk showed that prochloraz was transferred to the milk. In males a significant increase of nipple retention was found, and the bulbourethral gland weight was decreased, whereas other reproductive organs were unaffected. In addition cytochrome P450 (CYP)1A activities in livers were induced by prochloraz, possibly as a result of Ah receptor activation. Behavioral studies showed that the activity level and sweet preference of adult males were significantly increased. Overall these results strongly indicate that prochloraz feminizes the male offspring after perinatal exposure, and that these effects are due, at least in part, to diminished fetal steroidogenesis.

The 6-a-day study: effects of fruit and vegetables on markers of oxidative stress and antioxidative defense in healthy nonsmokers.

BACKGROUND: Fruit and vegetables contain both nutritive and nonnutritive factors that might contribute to redox (antioxidant and prooxidant) actions. OBJECTIVE: We investigated the relative influence of nutritive and nonnutritive factors in fruit and vegetables on oxidative damage and enzymatic defense. DESIGN: A 25-d intervention study with complete control of dietary intake was performed in 43 healthy male and female nonsmokers who were randomly assigned to 1 of 3 groups. In addition to a basic diet devoid of fruit and vegetables, the fruit and vegetables (Fruveg) group received 600 g fruit and vegetables/d; the placebo group received a placebo pill, and the supplement group received a vitamin pill designed to contain vitamins and minerals corresponding to those in 600 g fruit and vegetables. Biomarkers of oxidative damage to protein and lipids and of antioxidant nutrients and defense enzymes were determined before and during intervention. RESULTS: Plasma lipid oxidation lag times increased during intervention in the Fruveg and supplement groups, and the increase was significantly higher in the former. Plasma protein carbonyl formation at lysine residues also increased in both of these groups. Glutathione peroxidase activity increased in the Fruveg group only. Other markers of oxidative damage, oxidative capacity, or antioxidant defense were largely unaffected by the intervention. CONCLUSIONS: Fruit and vegetables increase erythrocyte glutathione peroxidase activity and resistance of plasma lipoproteins to oxidation more efficiently than do the vitamins and minerals that fruit and vegetables are known to contain. Plasma protein carbonyl formation at lysine residues increases because of the vitamins and minerals in fruit and vegetables.

The citrus-derived flavonoid naringenin exerts uterotrophic effects in female mice at human relevant doses.

Gavage administration of the citrus flavonoid naringenin, 3',4,5,7-tetrahydroxyflavanon for 4 consecutive days, to immature female mice (postnatal day 17-20) at 4 or 100 mg/kg b.wt. significantly increased uterine weights 3 and 4 times, respectively. Analysis of uterine oestrogen receptor alpha revealed that naringenin significantly increased the cytosolic concentration of oestrogen receptor alpha, whereas in nuclei the oestrogen receptor alpha concentration was significantly decreased as compared to the solvent control. This was in contrast to the positive control 17 beta-oestradiolacetate which acted as a true oestrogen by increasing the concentration of both total and nuclear oestrogen receptor alpha. Both naringenin and 17 beta-oestradiolacetate, however, significantly, induced nuclear oestrogen receptor alpha in the liver, suggesting a tissue specific effect of naringenin on oestrogen receptor alpha distribution. In order to investigate the tissue levels at which the uterotrophic effect was observed, the distribution of an oral dose of tritiated naringenin (4 mg/kg) was investigated in 3-week-old female mice. The radioactivity content (ng naringenin equivalents/g tissue) was found to be highest in the gastrointestinal-tract, followed by the kidneys and liver. Uterus and ovaries were also found to contain relatively high and approximately equal amounts of naringenin. The concentration of naringenin in uterus and ovaries was found to be ten times higher as compared to the mammary tissue. The urinary excretion of more than 25% of the administered dose, within 8 hr after dosing indicated that naringenin is absorbed extensively in mice. The plasma concentration of 0.5 microM found in the present study is similar to the peak plasma concentration of naringenin (0.6 microM) observed in man following ingestion of 400-760 ml of orange juice (Erlund et al. 2001). This could be taken to suggests that ingestion of orange juice and other citrus fruits and juices may give rise to sufficiently high tissue levels of naringenin in man to exert a biological effect.

Effects of dietary antioxidants and 2-amino-3-methylimidazo[4,5-f]- quinoline (IQ) on preneoplastic lesions and on oxidative damage, hormonal status, and detoxification capacity in the rat.

The potential beneficial or adverse affect of prolonged dietary administration of moderate to high doses (1-100 mg/kg diet) of the antioxidants, lycopene, quercetin and resveratrol or a mixture of lycopene and quercetin was investigated in male F344 rats. Selected markers for toxicity and defense mechanisms were assayed in blood, liver and colon and the impact of the antioxidant administrations on putative preneoplastic changes in liver and colon was assessed. The dietary carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (200 mg/kg diet) served as a pro-oxidant, genotoxicity and general toxicity control. IQ increased the levels of protein and DNA oxidation products in plasma, the area of glutathione S-transferase-placental form positive (GST-P) foci in the liver as well as the number of colonic aberrant crypt foci (ACF). All antioxidants and the antioxidant combination significantly increased the level of lymphocytic DNA damage, to an extent comparable with the effect induced by IQ. In contrast to the control group where no GST-P foci were detected, GST-P foci were detected in animals exposed to quercetin, lycopene and the combination of the two. However, the increase in the volume of GST-P foci did not reach statistical significance. The present results indicate that moderate to high doses of common dietary antioxidants can damage lymphocyte DNA and induce low levels of preneoplastic liver lesions in experimental animals. Long-term exposure to moderate to high doses of antioxidants may thus via pro-oxidative mechanisms and non-oxidative mechanisms modulate carcinogenesis.

Effect of in utero-administered coumestrol, equol, and organic selenium on biomarkers for phase 2 enzyme capacity and redox status.

The aim of the present study was to investigate the effect of in utero administration of coumestrol, equol, and selenium-enriched yeast on selected hepatic phase 2 enzymes, plasma hormone levels, and markers for redox status in plasma and red blood cells (RBCs). The test compounds were administered via the diet to pregnant Sprague-Dawley rats throughout gestation. Within 24 h following delivery dams and offspring were sacrificed, and blood, liver, and reproductive organs were sampled. Coumestrol, equol, and selenium-enriched yeast did not significantly affect hepatic glutathione S-transferase (GST), quinone reductase (QR), or RBC glutathione peroxidase (GPx) in the offspring, whereas significant increases in GST, QR, and GPx activities in dams were observed following administration of selenium-enriched yeast. The level of 17beta-estradiol in offspring from coumestrol-exposed dams was significantly increased compared with the control. The present results indicate that selenium-enriched yeast, coumestrol, and equol affect selected hepatic phase 2 enzymes and GPx in RBC in dams, whereas the offspring in general were refractive to the employed treatments. Further studies are warranted to investigate whether the observed in utero effects imposed by the selected plant compounds confer permanent alterations on the health status of the animal resulting in an altered resistance to cancer.

In vitro metabolism of genistein and tangeretin by human and murine cytochrome P450s.

Recombinant cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6 enzymes obtained from Escherichia coli and human liver microsomes samples were used to investigate the ability of human CYP enzymes to metabolize the two dietary flavonoids, genistein and tangeretin. Analysis of the metabolic profile from incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3'-hydroxylated metabolite of genistein. The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4'-hydroxy-5,6,7,8-tetramethoxyflavone and 5,6-dihydroxy-4',7,8-trimethoxyflavone, previously identified in rat urine in our laboratory. By comparison with UV spectra and LC/MS fragmentation patterns of previously obtained standards, the remaining metabolites eluting after 14, 17 and 20 min. were found to be demethylated at the 4',7-, 4',6-positions or hydroxylated at the 3'- and demethylated at the 4'-positions, respectively. Metabolism of tangeretin by recombinant CYP1A2, 3A4, 2D6 and 2C9 resulted in metabolic profiles that qualitatively were identical to those observed in the human microsomes. Inclusion of the CYP1A2 inhibitor fluvoxamine in the incubation mixture with human liver microsomes resulted in potent inhibition of tangeretin and genistein metabolism. Other isozymes-selective CYP inhibitors had only minor effects on tangeretin or genistein metabolism. Overall the presented observations suggest major involvement of CYP1A2 in the hepatic metabolism of these two flavonoids.

Effects of commonly consumed fruit juices and carbohydrates on redox status and anticancer biomarkers in female rats.

Administration of apple juice, black currant juice, or a 1:1 combination of the two juices significantly decreased the level of the lipid peroxidation biomarker malondialdehyde in plasma of female rats, whereas the protein oxidation biomarker 2-amino-adipic semialdehyde, was significantly increased following administration of orange juice, black currant juice, or the 1:1 combination of apple and black currant juice. A significant increase in 2-amino-adipic semialdehyde was also observed in control rats given sucrose, fructose, and glucose in the drinking water at concentrations approximating the average carbohydrate levels in the employed fruit juices. None of the fruit juices were found to affect the activities of antioxidant enzymes in red blood cells or hepatic glutathione S-transferase. Hepatic quinone reductase activity, on the other hand, was significantly increased by grapefruit juice, apple juice, and black currant juice. The total daily intake of a selected subset of flavonoid aglycones ranged from 0.2 to 4.3 mg, and quercetin was found to be a minor constituent of all the juices investigated. In a parallel study, rats were fed quercetin at doses ranging from 0.001 to 10 g/kg of diet. However, no effects were observed on hepatic glutathione S-transferase or quinone reductase activities, plasma redox status, or the activity of red blood cell antioxidant enzymes. Overall, the results of the present study suggest that commonly consumed fruit juices can alter lipid and protein oxidation biomarkers in the blood as well as hepatic quinone reductase activity, and that quercetin may not be the major active principle. The observation that natural carbohydrates are capable of mediating oxidative stress in vivo warrants further studies due to the central role refined and unrefined carbohydrates play in human nutrition.