Retinoid Analogues Research Articles

Pharmaceutical xenobiotics inducing thymus cellular apoptosis after retinoids administration during pregnancy. Experimental transmission electron microscopical study on balb/c mice

Event Abstract Back to Event Pharmaceutical xenobiotics inducing thymus cellular apoptosis after retinoids administration during pregnancy. Experimental transmission electron microscopical study on balb/c mice Emmanouel Nikoloussis1 and Elpida-Niki Emmanouil-Nikolousi1* 1 Aristotle University of Thessaloniki, Laboratory of Histology-Embryology and Anthropology, Greece Introduction. Thymus is an essential immunoregulatory organ, especially during pregnancy. Thymocytes can be induced to undergo apoptotic cell death by activation through the T-cell receptor (TCR). All-trans-RA is the high-affinity ligand for retinoic acid receptors ( RARs ),For this study we have identified the incidence of thymic cellular apoptosis during pregnancy after administration of all-trans and 13-cis retinoic acid. Methods. For this study we have treated pregnant female Balb/c mice 10 weeks old at gestational days 8th, 9th and 10th with the following substances: All-trans Retinoic-acid, 13-cis Retinoic acid and Corn Oil. Retinoid Analogues were diluted in corn oil and corn oil itself, were given by gastric intratubation to pregnant mice. Pregnant animal treatment was arranged as follows: Group 1: All-trans Retinoic acid 50 mg/k.b.w.into 0.5 gr corn oil; Group 2: 13-Cis Retinoic acid 50mg/k.b.w.into 0.5 gr corn oil; Group 3: Corn Oil 0.5 gr at all the corresponded with all-trans and 13-cis retinoic acid gestational days; Group 4: Untreated pregnant control. Pregnant animals were sacrificed on 19th gestational day. The thymus from each pregnant mouse was removed and embedded under routine procedures for observations at Transmission Electron Microscope (TEM). Results. Our findings showed normal ultrastructural cell architecture and regular type of apoptosis at the thymus cell population of the corn oil treated group and the untreated group of animals. Ultrastructural observations from thymus cell parenchyma from pregnant mice treated with all-trans R.A. showed severe pathological effects at the thymic parenchyma and increased apoptotic activity, accumulation of large secondary lysosomes and apoptotic bodies within the thymus dendritic cells. Conclusion. Apoptosis is a key feature in the physiological and pathological regulation of the immune system and thymus is one of the master glands regulating this system. Numerous compounds capable of influencing apoptosis are known, but their exact mechanisms of action are only in part understood. The majority of the known drugs does not affect apoptosis selectively, but alter the function of the whole organism; therefore considerable side-effects related to the application of these compounds may appear. Our results suggest a complex interaction between the various cell population consisting thymus and the administration of all-trans retinoic acid during pregnancy. Our knowledge will, presumably, be extended in the future and that could lead to the safer clinical application of pharmaceutical xenobiotic compounds as retinoids during pregnancy.

Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability ( P < 0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P < 0.05). The MeBo cells exhibited some inhibition with these natural ligands ( P < 0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects ( P < 0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability ( P < 0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability ( P < 0.05) and inhibited BME-UV1 cell viability ( P < 0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type ( P < 0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

Rexinoid-induced Expression of IGFBP-6 Requires RARβ-dependent Permissive Cooperation of Retinoid Receptors and AP-1

The synthetic rexinoid bexarotene (Targretin, LGD1069) inhibits the formation of both estrogen receptor-negative and estrogen receptor-positive breast cancer in preclinical models and controls the expression of growth-regulatory biomarkers, such as IGFBP-6 (insulin-like growth factor-binding protein 6), RARbeta, or cyclin D1. In this study, we identified a classical retinoic acid-responsive element in the first intron in the IGFBP-6 gene adjacent to a consensus AP-1 binding site, both elements essential for rexinoid-induced expression of IGFBP-6. In chromatin binding experiments, bexarotene increased the occupancy of the identified enhancer element by RXRalpha, RARbeta, cJun, cFos, and p300. In normal mammary epithelial cells and T47D breast cancer cells, small interfering RNA-mediated knockdown of all RXR isoforms or RARbeta, but not RARalpha or RARgamma alone, blocked the induction of IGFBP-6 by bexarotene. Simultaneous knockdown of RARalpha and RARgamma abrogated both the induction of RARbeta and the up-regulation and secretion of IGFBP-6. The suppression of either RARbeta or cJun by small interfering RNA blocked the recruitment of RXRalpha and cJun to the enhancer. These results demonstrate a novel cooperative interaction between retinoid receptors and AP-1 orchestrated by RARbeta and highlight a novel mechanism by which RARbeta can mediate the cancer-preventive effects of rexinoids.

Proteomic profiling of the stem cell response to retinoic acid and synthetic retinoid analogues: identification of major retinoid-inducible proteins

The natural retinoid, all-trans retinoic acid (ATRA), is widely used to direct the in vitro differentiation of stem cells. However, substantial degradation and isomerisation of ATRA in response to UV-vis light has serious implications with regard to experimental reproducibility and standardisation. We present the novel application of proteomic biomarker profiling technology to stem cell lysates to rapidly compare the differentiation effects of ATRA with those of two stable synthetic retinoid analogues, EC19 and EC23, which have both been shown to induce differentiation in the embryonal carcinoma cell line TERA2.cl.SP12. MALDI-TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry) protein profiles support previous findings into the functional relationships between these compounds in the TERA2.cl.SP12 line. Subsequent analysis of protein peak data enabled the semi-quantitative comparison of individual retinoid-responsive proteins. We have used ion exchange chromatographic protein separation to enrich for retinoid-inducible proteins, thereby facilitating their identification from SDS-PAGE gels. The cellular retinoid-responsive proteins CRABP-I, CRABP-II, and CRBP-I were up-regulated in response to ATRA and EC23, indicating a bona fide retinoid pathway response to the synthetic compound. In addition, the actin filament regulatory protein profilin-1 and the microtubule regulator stathmin were also elevated following treatment with both ATRA and EC23. The up-regulation of profilin-1 and stathmin associated with retinoid-induced neural differentiation correlates with their known roles in cytoskeletal reorganisation during axonal development. Immunological analysis via western blotting confirmed the identification of CRABP-I, profilin-1 and stathmin, and supported their observed regulation in response to the retinoid treatments.

The Role of Retinoids in the Adult Nervous System and their Therapeutic Potential

The mode of action of retinoids in relation to their activity in the adult central nervous system and the potential of synthetic retinoid analogues is reviewed. Investigation into the activity of such molecules will further our understanding of the retinoid pathway during nervous system development and in various neurological disease states.

A phase I trial of bexarotene and docetaxel in patients with advanced solid tumors

14512 Background: A phase I trial of bexarotene and docetaxel in patients with advanced solid tumors. Retinoids play critical roles in normal development and physiology by modulating cell growth, division, reproduction, differentiation, and immune function. Bexarotene (Bex) is a novel synthetic retinoid analogue binding preferentially to members of the RXR subclass of receptors. In a phase I trial of single agent Bex, patients with non-small cell lung cancer (NSCLC) had prolonged stabilization of disease and survival. Retinoids enhance docetaxel (D)induced apoptosis by causing down-regulation of Bcl-2, activation of RAR-gamma and altered expression Bcl-2/Bax. Methods: This Phase 1 study in patients (pts) with advanced neoplasms evaluated the safety and tolerability of fixed dose oral Bex at 400mg/m2 daily with escalating doses of D weekly for 3/4 weeks at 3 dose levels, 25, 30 & 35mg/m2, for up to 6 cycles. In pts exhibiting response or disease stabilization, treatment with Bex was continued until disease progression or intolerable toxicity. Cohorts consisted of 3 pts and were expanded to 6 pts if dose- limiting toxicity (DLT) was noted. Results: 19 pts were enrolled, including NSCLC (8), head & neck (2) esophageal (2) colorectal (2) cancers. 7 pts were treated at 25mg/m2, 6 at 30mg/m2 and 6 at 35mg/m2 of D. The MTD was D at 30mg/m2 with 400mg/m2of daily Bex. Hypothyroidism, hypertriglyceridemia, and fatigue were common.Two pts experienced grade 3 fatigue, and 1 each experienced grade 3 hypertriglyceridemia and hypothyroidism. Of 16 patients assessable for response, and 7 had stable disease for at least 2 cycles. One pt with NSCLC had a partial response that persisted for 8 cycles. 3 pts developed pulmonary toxicity (2 with radiation recall pneumonitis), 2 withdrew consent due to grade 3 fatigue. One pt died due to acute pulmonary hypertension secondary to pulmonary tumor emboli noted at autopsy. 5 pts are presently alive, two longest with NSCLC 35 & 22 months. Conclusions: MTD of D was 30mg/m2 in combination with daily dose Bex 400mg/m2. Care should be taken in patients who have been previously irradiated. A phase II trial of this combination at MTD as second line therapy in NSCLC is planned and correlative studies will be added. No significant financial relationships to disclose.

Synthesis and evaluation of synthetic retinoid derivatives as inducers of stem cell differentiation

All-trans-retinoic acid (ATRA) and its associated analogues are important mediators of cell differentiation and function during the development of the nervous system. It is well known that ATRA can induce the differentiation of neural tissues from human pluripotent stem cells. However, it is not always appreciated that ATRA is highly susceptible to isomerisation when in solution, which can influence the effective concentration of ATRA and subsequently its biological activity. To address this source of variability, synthetic retinoid analogues have been designed and synthesised that retain stability during use and maintain biological function in comparison to ATRA. It is also shown that subtle modifications to the structure of the synthetic retinoid compound impacts significantly on biological activity, as when exposed to cultured human pluripotent stem cells, synthetic retinoid 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid, 4a (para-isomer), induces neural differentiation similarly to ATRA. In contrast, stem cells exposed to synthetic retinoid 3-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid, 4b (meta-isomer), produce very few neurons and large numbers of epithelial-like cells. This type of structure-activity-relationship information for such synthetic retinoid compounds will further the ability to design more targeted systems capable of mediating robust and reproducible tissue differentiation.

Antitumor activity of 4-( N-hydroxyphenyl)retinamide conjugated with poly( l-glutamic acid) against ovarian cancer xenografts

Objective. Natural and synthetic retinoids such as N-(4-hydroxyphenyl)retinamide (4HPR) have been used for prevention and treatment of a variety of cancers; however, relapse usually occurs after treatment is stopped. Furthermore, the retinoid analogues are insoluble in water, making it difficult for systemic administration. The purpose of this study was to develop and evaluate a water-soluble polymeric formulation of 4HPR that can release 4HPR over a period of a few days. Methods. 4HPR was bound to a synthetic polyamino acid poly( l-glutamic acid) (PG). PG–4HPR was evaluated for its release kinetics and in vitro anti-proliferative and in vivo antitumor activities against ovarian cancer cell lines. Results. The release profile of PG–4HPR in phosphate buffered saline at 37 °C followed a first order kinetic, with a rate constant of 8.8 × 10 − 3 h − 1 . Approximately 60% of 4HPR was released over a period of 100 h. In vitro, both 4HPR and PG–4HPR inhibited proliferation of three ovarian cancer cells lines (SKOV3, OVCA420, and OVCA433) and an immortalized human ovarian epithelium cell line (IOSE) in a time- and dose-dependent manner. Increasing the exposure time of SKOV3 cells to both agents from 1 to 5 days resulted in an increased apoptotic response. In vivo, PG–4HPR demonstrated significantly enhanced antitumor activities compared to 4HPR in both early treatment and later treatment protocols. Treatments with PG–4HPR suppressed the expression of VEGF and reduced blood flow into the tumor. Conclusions. PG–4HPR may have potential applications in the prevention and therapy of ovarian cancers.

Chemoprevention of liver carcinogenesis with retinoids: Basic and clinical aspects

The strategy to prevent liver carcinogenesis consists of: (i) antiviral modalities such as vaccination, lamivudin, and interferon; (ii) anti-inflammatory modality; and (iii) chemoprevention using such compounds as retinoid analog and vitamin K. Cancer chemoprevention is defined as an approach where natural or synthetic chemical compound works to arrest or reverse premalignant cells by using physiological pathways. As a consequence, such a clone of premalignant cells is eradicated (clonal deletion) by differentiation induction or apoptosis, and thus the process toward the development of clinically detectable cancer is disrupted. A particularly effective candidate target of chemoprevention in liver diseases is an advanced stage of chronic hepatitis, that is supposed to contain transformed hepatocyte clone(s); that is, primary prevention from liver cirrhosis and prevention of recurrent and second primary hepatocellular carcinoma following the treatment of the initial cancer. Retinoid is a collective term of vitamin A analog that binds to nuclear retinoid receptors;retinoic acid receptors (RAR) and retinoid X receptors (RXR). After ligand coupling, these receptors form homo- or heterodimers, bind to the response element (RARE or RXRE) upstream of the target gene, and regulate the gene expression as a transcriptional factor. Biological phenotypes of such transcriptional regulation by retinoid include cellular differentiation, tissue morphogenesis, and programmed cell death (apoptosis). Due to these functions, retinoid analogs are clinically tried to prevent/treat carcinoma in a wide variety of organs including head and neck cancer, uterine cervical cancer, certain leukemia and liver cancer. In this article, clinical trials of retinoid analog to inhibit second primary hepatoma, supposed molecular mechanism of the action of the compound, and aberrant metabolism of RXR and its role in liver carcinogenesis are briefly reviewed.

Hypertriglyceridemia presenting as “pink blood” and elevated hemoglobin level

American Journal of HematologyVolume 83, Issue 3 p. 253-253 Images in HematologyFree Access Hypertriglyceridemia presenting as “pink blood” and elevated hemoglobin level Donald E. Tsai, Corresponding Author Donald E. Tsai detsai@mail.med.upenn.edu Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaUniversity of Pennsylvania Medical Center, 16 Penn Tower, 3400 Spruce Street, Philadelphia, PA 19104Search for more papers by this authorAnthony Mato, Anthony Mato Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaSearch for more papers by this authorDavid L. Porter, David L. Porter Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaSearch for more papers by this authorDan T. Vogl, Dan T. Vogl Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaSearch for more papers by this author Donald E. Tsai, Corresponding Author Donald E. Tsai detsai@mail.med.upenn.edu Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaUniversity of Pennsylvania Medical Center, 16 Penn Tower, 3400 Spruce Street, Philadelphia, PA 19104Search for more papers by this authorAnthony Mato, Anthony Mato Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaSearch for more papers by this authorDavid L. Porter, David L. Porter Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaSearch for more papers by this authorDan T. Vogl, Dan T. Vogl Abramson Cancer Center, University of Pennsylvania Medical Center, Philadelphia, PennsylvaniaSearch for more papers by this author First published: 27 August 2007 https://doi.org/10.1002/ajh.21054AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Duvic M,Hymes K,Heald P, et al. Bexarotene is effective and safe for treatment of refractory advanced-stage cutaneous T-cell lymphoma: Multinational phase II–III trial results. J Clin Oncol 2001; 19: 2456– 2471. 2 Tsai DE,Aqui NA,Vogl RD, et al. Successful treatment of T-cell posttransplant lymphoproliferative disorder with the retinoid analog bexarotene. Am J Transplant 2005; 5: 2070– 2073. 3 Shah PC,Patel AR,Rao KRP. Hyperlipidemia and spuriously elevated hemoglobin values. Ann Int Med 1975; 82: 382– 383. Volume83, Issue3March 2008Pages 253-253 ReferencesRelatedInformation

Delivery of Retinoid-Based Therapies To Target Tissues

Through its various metabolites, vitamin A controls essential physiological functions. Both naturally occurring metabolites and novel retinoid analogues have shown effectiveness in many clinical settings that include skin diseases and cancer, and in animal models of human conditions affecting vision. In this review, we analyze several potential retinoid-based therapies from the point of view of drug metabolism and transport to target tissues. We focus on the endogenous factors that affect the absorption, transport, and metabolism of retinoids by taking into account data obtained from the analysis of animal models that lack the enzymes or proteins involved in the storage and absorption of retinoids. We also discuss findings of toxicity associated with retinoids in an effort to improve the outcome of retinoid-based therapies. In this context, we review evidence that esterification of retinol and retinol-based drugs within target tissues provides one of the most efficient means to improve the absorption and to reduce the toxicity associated with pharmacological doses of retinoids. Future retinoid-based therapeutic strategies could involve targeted delivery mechanisms leading to lower toxicity and improved effectiveness of retinoids.

Targeting homeostatic mechanisms of endoplasmic reticulum stress to increase susceptibility of cancer cells to fenretinide-induced apoptosis: the role of stress proteins ERdj5 and ERp57

Endoplasmic reticulum (ER) malfunction, leading to ER stress, can be a consequence of genome instability and hypoxic tissue environments. Cancer cells survive by acquiring or enhancing survival mechanisms to counter the effects of ER stress and these homeostatic responses may be new therapeutic targets. Understanding the links between ER stress and apoptosis may be approached using drugs specifically to target ER stress responses in cancer cells. The retinoid analogue fenretinide [N-(4-hydroxyphenyl) retinamide] is a new cancer preventive and chemotherapeutic drug, that induces apoptosis of some cancer cell types via oxidative stress, accompanied by induction of an ER stress-related transcription factor, GADD153. The aim of this study was to test the hypothesis that fenretinide induces ER stress in neuroectodermal tumour cells, and to elucidate the role of ER stress responses in fenretinide-induced apoptosis. The ER stress genes ERdj5, ERp57, GRP78, calreticulin and calnexin were induced in neuroectodermal tumour cells by fenretinide. In contrast to the apoptosis-inducing chemotherapeutic drugs vincristine and temozolomide, fenretinide induced the phosphorylation of eIF2α, expression of ATF4 and splicing of XBP-1 mRNA, events that define ER stress. In these respects, fenretinide displayed properties similar to the ER stress inducer thapsigargin. ER stress responses were inhibited by antioxidant treatment. Knockdown of ERp57 or ERdj5 by RNA interference in these cells increased the apoptotic response to fenretinide. These data suggest that downregulating homeostatic ER stress responses may enhance apoptosis induced by oxidative stress-inducing drugs acting through the ER stress pathway. Therefore, ER-resident proteins such as ERdj5 and ERp57 may represent novel chemotherapeutic targets.

Editorial: Tissue Engineering: Perspectives, Challenges, and Future Directions

Editorial: Tissue Engineering: Perspectives, Challenges, and Future Directions

Fenretinide (4HPR) Inhibits Growth of Myeloma Cells in Their Microenvironment and Is a Potent Inhibitor of Angiogenesis and Osteoclastogenesis.

Fenretinide (4HPR) is a relatively safe neoclassical retinoid analog that inhibits growth of various tumors through increased intracellular ceramide and ROS, induction of tumor cell apoptosis and inhibition of angiogenesis. 4HPR has been successfully tested as a chemopreventive and chemotherapeutic agent in clinical trials on various malignancies. In contrast to retinoic acid, 4HPR induces cell apoptosis rather than differentiation and shows synergistic responses with chemotherapeutic drugs in different tumor cell types. The biological effect and therapeutic value in multiple myeloma (MM) has not been investigated. The aim of this study was to investigate the anti-MM effect and mechanism of action of 4HPR using 3 stroma-dependent and 2 stroma-independent MM cell lines established in our laboratory, CD138-selected primary MM cells and co-culture systems of these cells with human osteoclasts and mesenchymal stem cells (MSCs) as previously described (Yaccoby et al., Cancer Res 2004). MM cell apoptosis detected by annexin V flow cytometry and TUNNEL, tumor growth by MTT assay, changes in caspase 3, 8 and 9 activity using Western blotting and ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay. 4HPR inhibits growth of all tested MM cells in a dose- and time-dependent manner. The IC50 after 48 hrs in serum-containing media was 10 μM using MTT assay. 4HPR (3 μM) increased percent of apoptotic MM cells by 2.5±0.4 folds (p<0.01). Co-culture of these cell lines with osteoclasts only partially protected MM cells from the proapoptotic effect of this drug. Furthermore, 4HPR also induced apoptosis of primary CD138-selected MM cells co-cultured with osteoclasts or MSCs, and inhibited growth of bortezomib-resistant MM cell lines. In contrast, 4HPR had only minimal cytotoxic effect on blood mononuclear cells and MSCs. The proapoptotic effect of 4HPR involved increased level of ROS by 2.55±0.67 folds in MM cells (p<0.01). We also detected reduced levels of procaspase and increased cleaved caspase 8, 9 and 3 within 24 hrs of incubation with this drug. Sphingosine-1 phosphate (S1P) partially protected MM cells from 4HPR-induced apoptosis suggesting that, as reported for other tumors, anti-MM mechanism of this drug involved increased intracellular ceramide. 4HPR significantly inhibited tube formation by HUVEC in a matrigel assay (p<0.0001), confirming its anti-angiogenic potential. This drug also effectively prevented formation of multinucleated osteoclasts in culture of human osteoclast precursors with RANKL and M-CSF (p<0.0001). Furthermore, mature osteoclasts viability as assessed by MTT assays was reduced following incubation with 3 μM 4HPR (p<0.0001). We conclude that 4HPR is a potent anti-MM agent, affecting growth of MM cells in their microenvironment directly through induction of apoptosis in mechanisms involving ROS, caspase and possibly ceramide, and indirectly through inhibition of angiogenesis and osteoclastogenesis. Our data also suggests that S1P, which is highly produced by activated platelets, is an important survival factor for MM cells. Study is underway to test anti-MM efficacy of 4HPR in the SCID-hu model for primary myeloma.

Stereoselective Synthesis of Trienoic Acids: Synthesis of Retinoic Acids and Analogues

Stereoselective construction of conjugated trienoic acids was achieved through two successive Stille reactions, the first step consisting of the coupling of (E)-1,2-bis(tributylstannyl)ethene and tributylstannyl (Z)- or (E)-3-iodoalk-2-enoates. Two different routes were used for the second step: (1) cross-coupling of the stannyldienoic acid reagents and vinyl iodides, or (2) cross-coupling of vinylstannane reagents and the tributylstannyl 5-iodopenta-2,4-dienoates generated by iododestannylation of stannyldienes. Vinyl-stannanes synthesized by stannylmetalation of the Negishi dienyne derived from β- or α-ionone and safranal thus provided access to stereodefined retinoic acids. Some retinoid and yne analogues were also prepared by Sonogashira coupling.

Chemoprevention of Skin Carcinogenesis by Phenylretinamides: Retinoid Receptor–Independent Tumor Suppression

Fenretinide [N-(4-hydroxyphenyl)retinamide or 4-HPR] is a synthetic retinoid analogue with antitumor and chemopreventive activities. N-(4-Methoxyphenyl)retinamide (4-MPR) is the most abundant metabolite of 4-HPR detected in human serum following 4-HPR therapy. We have shown in in vitro studies that 4-HPR and 4-MPR can act independent of the classic nuclear retinoid receptor pathway and that 4-HPR, but not 4-MPR, can also activate nuclear retinoid receptors. In this study, we have compared the chemopreventive effects of topically applied 4-HPR and 4-MPR with the primary biologically active retinoid, all-trans retinoic acid (ATRA), in vivo in the mouse skin two-stage chemical carcinogenesis model. All three retinoids suppressed tumor formation but the effect of 4-HPR and 4-MPR, and not of ATRA, was sustained after their discontinuation. The tumor-suppressive effects of 4-HPR and 4-MPR were quantitatively and qualitatively similar, suggesting that the two may be acting through the same retinoid receptor-independent mechanism(s). We further explored this effect in vitro by analyzing primary cultures of mouse keratinocytes treated with the same retinoids. All three could induce apoptosis with a 48-hour treatment and only ATRA and 4-HPR induced an accumulation of cells in the G1 phase of the cell cycle. This finding is consistent with our previous results showing that the effects of phenylretinamides on the cell cycle are retinoid receptor dependent whereas apoptosis induction is not. A microarray-based comparison of gene expression profiles for mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) alone and TPA + 4-HPR or TPA + 4-MPR reveals a high degree of coincidence between the genes regulated by the two phenylretinamides. We propose that 4-HPR may exert therapeutic and chemopreventive effects by acting primarily through a retinoid receptor-independent mechanism(s) and that 4-MPR may contribute to the therapeutic effect of 4-HPR by acting through the same retinoid receptor-independent mechanism(s).

Defining the Retinoid Binding Site in the Rod Cyclic Nucleotide-gated Channel

Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521–531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, β-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid–channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues ∼15 Å away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.

Synthesis of New 9‐Methylene Analogs of Retinoids

We report herein a series of syntheses that provides diverse structural modifications of the side‐chain of retinoids to obtain new compounds with potential use in anticancer therapy. Starting from a β‐methylenealdehyde synthon, we have synthesized a series of new 9‐methylene‐13‐desmethyl‐14‐methyl analogs and a series of 9‐methylene‐11‐desmethyl trienic homologs. For the first series, the condensation of the C‐15 β‐methylenealdehyde with the anion of ethyl 4‐(diethoxyphosphoryl)‐2E‐methylbut‐2‐enoate led to the 7E,11E,13E‐ester. This gave the desired aldehyde by a reduction into the corresponding alcohol (DIBAL‐H) and subsequent oxidation by MnO2. For the second series, the reaction of the C‐15 β‐methylenealdehyde with the anions of ethyl diethoxyphosphorylacetate, diethyl cyanomethylphosphonate, or diethyl 2‐oxopropylphosphonate led to the C‐17 ester, the C‐17 nitrile, and the C‐18 ketone, respectively.

Bexarotene improves TTP in untreated, advanced NSCLC, when given in combination with carboplatin/paclitaxel

7270 Background: Bexarotene, a specific synthetic retinoid analogue, binds to the α, β, and γ subclass of RXR, providing therapeutic specificity and reduced toxicities in pts with RXR-expressing tumors. Initial phase-I/II clinical trials in NSCLC showed that bexarotene added to chemotherapy prolonged stabilization of disease (TTP) and 1, 2, and 3 yr survival. Methods: Stage IIIB with pleural effusion & Stage IV chemo-naïve patients, ECOG PS 0–2, were enrolled on study and treated with carboplatin IV AUC-6 d1 and paclitaxel IV 100mg/m2 d1, 8, and 15, every 28-d for 4 cycles. Pts were randomized using a 1:1 design to bexarotene PO 400mg/m2/d either concurrent (C) from Day 1 or sequential (S) at the completion of chemo, for up to a year. Results: From a planned total of 60 patients, 48 have been enrolled thus far; median age 62.3 (range 41–86), 43 Caucasian, 41 TNM Stage IV, 33 males, 34 ECOG PS 1. To date, 44 pts are evaluable for efficacy and toxicity based on ITT; 35 were evaluated for RR: 15 (42.8%) achieved PR (C: 7 and S: 8), 15 (42.8%) exhibited SD (C: 8 and S: 7), and 5 (14.3%) had PD (C: 2 and S: 3), during the first 112 days (C1–4 chemo). TTP analysis was done in 42 pts: 19 pts showed an overall TTP of 152 days (5.06 mo) (C=10: 148.3 days and S=9: 155.5 days); in 23 pts the TTP has not been reached after a median F/U of 79 days (range 10–203). The overall 1yr S was 58.8% with no significant difference between treatment arms (p=0.7). The treatment was well tolerated; overall, AEs were reported in 48% of pts in the S arm vs. 51% in the C arm. The incidence of Gr 3–4 AEs, regardless of the treatment arm, was < 5%. There were no treatment-associated deaths. Conclusions: so far, data suggests a comparable ORR and a potential improvement in TTP, when bexarotene is added to carboplatin/paclitaxel, compared with chemo alone. Toxicity is easily managed. Updated efficacy and toxicity data will be presented at the meeting. No significant financial relationships to disclose.

Acquisition of breast tissue in a biomarker modulation study using bexarotene in women at high risk of breast cancer

1002 Background: Bexarotene is a synthetic retinoid analogue that binds to members of the RXR subclass of receptors. We have previously demonstrated that this RXR-selective retinoid prevents ER-negative breast cancer in preclinical animal models. We are now testing this agent in a randomized, placebo-controlled Phase II cancer prevention trial in women at high risk of breast cancer. The primary endpoint of this trial is change in breast epithelial cell proliferation measured by Ki67 immunohistochemical staining after drug treatment. Secondary endpoints are measurement of other retinoid-regulated biomarkers (apoptosis, ER, PR, retinoid regulated genes). Methods: Women at high risk of breast cancer (defined by having ≥ 10% chance of a BRCA1 or 2 mutation) were enrolled in this multi-institutional trial. Women received study drug (placebo or bexarotene at 200mg/m2) for 28 days. Non-directed breast core needle biopsies were taken before and after the study medication. Biomarker analysis will be performed using these samples. Results: 47 of planned 100 subjects have been enrolled; 37 have taken the study drug or placebo. Median age: 42 years (range: 20–82). Median risk of carrying a BRCA1/2 mutation: 30% (range: 11–100%) by the BRCAPro, Couch, or Myriad models. Genetic testing for BRCA1 or 2 mutations was done for all subjects. Seven subjects (16.6%) were found to have a mutation in either BRCA1 or BRCA 2. 37 women had baseline breast biopsies. Of 69 samples, 52 (75%) were satisfactory for biomarker analysis (defined by the presence of epithelial cells on H&E). Epithelial cell adequacy rates increased over time. 30 patients had pre- and post-treatment biopsies. 23/30 (77%) of the day 1 and 23/30 (77%) of the day 28 biopsies were satisfactory; 19 of 30 (63%) of the paired samples are satisfactory and available for analysis. Conclusions: Current results from this study demonstrate that accrual of high-risk women to biomarker modulation trials of novel agents is feasible. Adequate tissue for analysis of many biomarkers has been obtained using core needle biopsy. Once accrual is complete we will determine whether bexarotene suppresses breast epithelial cell growth in high-risk women. Supported by NIH grant U19CA086809. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ligand NCI, NIH