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Substrates of a major drug-metabolizing enzyme CYP2D6 display increased elimination during pregnancy, but the underlying mechanisms are unknown in part due to a lack of experimental models. Here, we introduce CYP2D6-humanized (Tg-CYP2D6) mice as an animal model where hepatic CYP2D6 expression is increased during pregnancy. In the mouse livers, expression of a known positive regulator of CYP2D6, hepatocyte nuclear factor 4α (HNF4α), did not change during pregnancy. However, HNF4α recruitment to CYP2D6 promoter increased at term pregnancy, accompanied by repressed expression of small heterodimer partner (SHP). In HepG2 cells, SHP repressed HNF4α transactivation of CYP2D6 promoter. In transgenic (Tg)-CYP2D6 mice, SHP knockdown led to a significant increase in CYP2D6 expression. Retinoic acid, an endogenous compound that induces SHP, exhibited decreased hepatic levels during pregnancy in Tg-CYP2D6 mice. Administration of all-trans-retinoic acid led to a significant decrease in the expression and activity of hepatic CYP2D6 in Tg-CYP2D6 mice. This study provides key insights into mechanisms underlying altered CYP2D6-mediated drug metabolism during pregnancy, laying a foundation for improved drug therapy in pregnant women.

Background: Alitretinoin (9-cis-retinoic acid, Toctino(®) ) has been marketed recently for oral therapy for chronic hyperkeratotic hand eczema. As alitretinoin is highly lipophilic and metabolized mainly in the liver, it is currently considered to be contraindicated in patients with liver disease. However, the pharmacokinetics and metabolism of alitretinoin have not been studied in these patients., Objectives: To study the single-dose pharmacokinetics and metabolism of alitretinoin and its metabolites in patients with cirrhosis following oral administration., Methods: Eight patients with cirrhosis and eight matched volunteer healthy controls were given a single 30-mg oral dose of alitretinoin. Blood and urine samples were collected during the following 24-h study period. Samples were analysed for alitretinoin and for known metabolites using reverse-phase high-performance liquid chromatography. The pharmacokinetics were then evaluated using standard noncompartmental models., Results: No significant differences were found between healthy controls and patients with cirrhosis when analysing the pharmacokinetic parameters of alitretinoin and its metabolites. Thus, the mean half-lives of alitretinoin were 5·3 and 5·6 h (P = 0.733) and the oral clearances were 1·92 and 1·39 L h(-1) kg(-1) (P = 0·243) in the patient group and the healthy control group, respectively., Conclusions: The metabolism and pharmacokinetics of alitretinoin following oral administration of the recommended dose of 30 mg for the treatment of severe hand eczema were similar in patients with cirrhosis and in healthy controls. If indicated, alitretinoin can be used in these patients with careful and close monitoring., (© 2013 British Association of Dermatologists.)

Poly(amino acid)s are well-known as biodegradable and environmentally acceptable materials. In this study, a series of poly(L-aspartic acid)-b-poly(L-phenylalanine) (PAA-PPA) compounds with different degrees of polymerization were used to prepare copolymer micelles for a poorly water-soluble drug 4-amino-2-trifluoromethyl-phenyl retinate (ATPR, a novel all-trans retinoic acid derivative) and in vivo pharmacokinetics, biodistribution and antitumor efficacy of ATPR delivered by PAA-PPA micelles were evaluated. The area under the plasma concentration time curve AUC0→∞ of ATPR-loaded PAA20PPA20 micelles was 2.23 and 1.97 times higher than that of ATPR solution and ATPR CrmEL solution, respectively; In addition, the mean residence time (MRT) was increased 1.67 and 1.97-fold, respectively and the total body clearance (CL) was reduced 2.25 and 1.98-fold, respectively. The biodistribution study indicated that most of the ATPR in the ATPR-M group was distributed in the liver and there was delayed liver aggregation compared with the ATPR solution and ATPR CrmEL solution groups. Furthermore, the antitumor efficacy of ATPR-loaded PAA20PPA20 micelles was demonstrated in in vivo antitumor models involving mice inoculated with the human gastric cancer cell line SGC-7901. At the same dose of 7mg/kg, the ATPR-loaded micelles group demonstrated a better tumor growth inhibition and induced differentiation than the groups given ATPR solution and ATPR CrmEL solution. Therefore, the ATPR-loaded PAA-PPA micelles appear to be a potentially useful drug delivery system for ATPR and suitable for the chemotherapy of gastric cancer., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A sensitive, reliable and specific LC-MS-MS method was developed and validated for the identification and quantitation of all-trans retinoic acid (ATRA) in human plasma. Acitretin was used as the internal standard (IS). After liquid-liquid extraction of 500 μL plasma with methyl tert-butyl ether (MTBE), ATRA and the IS were chromatographed on a HyPURITY C18 column (150 mm×2.1 mm, 5 μm) with the column temperature set at 40 °C. The mobile phase was consisted of 40% phase A (MTBE-methanol-acetic acid, 50:50:0.5, v/v) and 60% phase B (water-methanol-acetic acid, 50:50:0.5, v/v) with a flow rate of 0.3 mL/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode via the positive electrospray ionization interface using the transition m/z 301.4→123.1 for ATRA and m/z 326.9→177.1 for IS, respectively. The calibration curve was linear over the range of 0.45-217.00 ng/mL (r≥0.999) with a lower limit of quantitation (LLOQ) of 0.45 ng/mL. The intra- and inter-day precisions values were below 8% relative standard deviation and the accuracy was from 98.98% to 106.19% in terms of relative error. The validated method was successfully applied in a bioequivalence study of ATRA in Chinese healthy volunteers.

In the present study, terpene composited lipid nanoparticles and lipid nanoparticles were developed and evaluated for dermal delivery of all-trans-retinoic acids (ATRA). Terpene composited lipid nanoparticles and lipid nanoparticles were investigated for size, size distribution, zeta potential, entrapment efficiency, photostability, and cytotoxicity. In vitro skin permeation of ATRA lipid formulations were also evaluated. To explore the ability of lipid nanocarriers to target the skin, the distribution of rhodamine B base in the skin was investigated using confocal laser scanning microscopy (CLSM). The results indicated that the physicochemical characteristics of terpene composited lipid nanoparticles influenced skin permeability. All lipid nanocarriers significantly protected ATRA from photodegradation and were non-toxic to normal human foreskin fibroblast cells in vitro. Solid lipid nanoparticles containing 10% limonene (10% L-SLN) had the highest ATRA skin permeability. Terpene composited SLN and nanostructured lipid carriers (NLC) showed higher epidermal permeation of rhodamine B across the skin based on CLSM image analysis. Our study suggests that terpene composited SLN and NLC can be potentially used as dermal drug delivery carriers for ATRA.

We synthesized functional retinoic acid (RA)-polyethyleneimine (PEI) complex nanoparticles. NH groups of branched PEI chains were electrostatically interacted with carboxyl groups of RA surfaces to form cationic RA-PEI complex nanoparticles. We observed that the average diameter of RA-PEI complex nanoparticles was approximately 70 nm and the morphology of complex nanoparticles was homogeneous circular shape. To confirm the synthesis of RA-PEI complex nanoparticles, we characterized complex nanoparticles using (1)H nuclear magnetic resonance (NMR), indicating that hydrophilic branched PEI chains were covered on hydrophobic RA surfaces. Furthermore, we demonstrated that pH enabled the control of amounts of RA released from RA-PEI complex nanoparticles, showing that RA exposed to acidic pH 5 was steadily released (∼76%) from complex nanoparticles, whereas RA was rapidly released (∼97%) at pH 7.4 on day 11. We also observed that RA-PEI complex nanoparticles induced embryonic stem (ES) cell-derived neuronal differentiation. Therefore, this RA-PEI complex nanoparticle is a potentially powerful tool for directing murine ES cell fate.

Purpose: All-trans retinoic acid (RA)-incorporated nanoparticles were prepared using deoxycholic acid-conjugated dextran (DexDA). Anticancer activity of RA-incorporated DexDA nanoparticles were tested in vitro and in vivo., Methods: RA-incorporated nanoparticles were prepared by dialysis. Antiproliferative and anti-invasive potential of RA-incorporated nanoparticles were studied using CT26 colorectal carcinoma cells., Results: RA-incorporated nanoparticles have small particle sizes of around 70-300 nm and spherical shapes. The higher drug-feeding ratio and higher substitution degree of deoxycholic acid in the conjugates resulted in higher drug contents, lower loading efficiency, and larger particle size. RA release rate became slower at higher drug contents and higher substitution degree of deoxycholic acid in the DexDA conjugates. The antiproliferation activity, anti-invasive activity, and matrix metalloproteinase 2 expression of RA-incorporated nanoparticles against CT26 cells in vitro was similar to RA. However, RA-incorporated nanoparticles had superior antimetastatic activity in an animal pulmonary metastatic model of CT26 cells compared to RA itself., Conclusion: RA-incorporated nanoparticles showed similar anticancer activity in vitro and superior antimetastatic activity in vivo in a pulmonary metastatic model of CT26 cells. We suggest that RA-incorporated nanoparticles are promising vehicles for efficient delivery of RA.

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the β-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.

Introduction: Microdialysis (MD) allows sampling of compounds in-vivo from tissues' interstitial fluid. However, molecules insoluble at physiological pH have usually extremely low recovery. The addition of albumin to the perfusate or the use of isotonic lipoemulsion improves recovery of these molecules although it requires a cleaning step before HPLC analysis. This study investigates the possibility of improving the MD recovery of compounds insoluble at physiological pH but soluble at a different pH. The probe is perfused with an isotonic solution adjusted to pH values at which the compound has maximum solubility. Ketoconazole (KTC), clotrimazole (CLT) and tretinoin (TTN) were selected as model drugs because they are almost insoluble at pH 7.4 but soluble at pH 4 for KTC and CTL; and at pH 9 for TTN., Methods: Linear microdialysis probes were used to collect KTC, CLT or TTN from a standard solution of the compounds. Probes were perfused with 0.01 M pH 7.4 isotonic buffer solution (1) without or (2) with 5% Bovine Serum Albumin (BSA); or (3) with 20% isotonic lipoemulsion; or (4) with 0.01 M pH 4 isotonic buffer solution for KTC and CLT or 0.01 M pH 9 isotonic buffer solution for TTN. The method was then tested in-vivo, in rabbit skin, to assess the skin tolerance to the non-physiological perfusates and to monitor KTC and TTN delivery from commercial cream products., Results: In-vitro, the optimized-pH perfusate increased MD recovery significantly (P<0.001): 6.9 (KTC), 8.3 (CLT), and 2.0 (TTN) times compared to the physiological pH and 1.4 and 1.2 compared to the BSA and lipoemulsion respectively. No evidence of irritation or edema was observed in-vivo. However, KTC and TTN were not detected in-vivo with any of the modified perfusate tested., Discussion: These findings show that the optimized-pH perfusate effectively increases the in-vitro microdialysis recovery of KTC, CLT and TTN and that it is well tolerated in-vivo. However, the compounds tested (KTC and TTN) could not be detected in-vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Acute promyelocytic leukemia (APL) is a rare form of acute myeloid leukemia with specific epidemiological, pathogenetic and clinical features. Its frequency varies widely among nations, with a decreased incidence among 'Nordic' origin populations. The molecular hallmark of the disease is the presence of a balanced reciprocal translocation resulting in the PML/RAR-α gene fusion, which represents the target of the all-trans retinoic acid (ATRA) therapy. The introduction of ATRA in conjunction with anthracyclines marked a turning point in the treatment of APL, previously associated with a significant morbidity and mortality. Nowadays the standard front-line therapy for pediatric APL includes ATRA in every phase of the treatment, resulting in a complete remission rate of 90-95%. Here we provide an overview of the role of ATRA in the treatment of pediatric APL, summarizing the most relevant clinical results of recent decades and investigating future therapeutic perspectives for children with APL.

Purpose: Novel retinoic acid metabolism blocking agent (RAMBA), VN/12-1, is a highly potent anti-cancer agent that induces autophagy. Its combination with autophagy inhibitor chloroquine (CHL) has been shown to synergistically enhance apoptosis in breast cancer cells. The purpose of this study was to determine the toxicity and pharmacokinetic profile of VN/12-1 and its combination with CHL., Methods: Preliminary toxicology of VN/12-1 was determined using female SCID mice (n = 4 for each group). ATRA was used for comparison. We selected four different doses of VN/12-1 and ATRA. Two of the doses were low and less frequent (2.5 and 5 mg/kg twice a week), and the remaining doses were high and more frequent (10 and 20 mg/kg every day). The dose of CHL was 50 mg/kg twice a week. For pharmacokinetic (PK) study, 20 mg/kg of VN/12-1 was injected subcutaneously (s.c.) into the mice, and their plasma was collected at various intervals (n = 2) and analyzed by HPLC., Results: The lower and less frequent doses of VN/12-1 and ATRA were found to be least toxic. However, high and more frequent doses of these compounds were toxic to the mice. PK results showed that VN/12-1 has a half-life of 6 h. The area under the curve (AUC) for VN/12-1 was 83.78 h μg/ml., Conclusions: VN/12-1 and ATRA are non-toxic when used as 5 mg/kg twice a week as single agents or in combination with CHL. The favorable PK properties of VN/12-1 can potentially be used for its further advanced pre-clinical and clinical development.

Amphiphilic low molecular weight heparin-all-trans-retinoid acid (LHR) conjugate, as a drug carrier for cancer therapy, was found to have markedly low toxicity and to form self-assembled nanoparticles for simultaneous delivery of paclitaxel (PTX) and all-trans-retinoid acid (ATRA) in our previous study. In the present study, PTX-loaded LHR nanoparticles were prepared and demonstrated a spherical shape with particle size of 108.9 nm. Cellular uptake analysis suggested rapid internalization and nuclear transport of LHR nanoparticles. In order to investigate the dynamic behaviors and targeting ability of LHR nanoparticles on tumor-bearing mice, near-infrared fluorescent (NIFR) dye DiR was encapsulated into the nanoparticles for ex vivo optical imaging. The results indicated that LHR nanoparticles could enhance the targeting and residence time in tumor site. Furthermore, in vivo biodistribution study also showed that the area under the plasma concentration time curve (AUC (0→inf)) values of PTX and ATRA for PTX-loaded LHR nanoparticles in tumor were 1.56 and 1.62-fold higher than those for PTX plus ATRA solution. Finally, PTX-loaded LHR nanoparticles demonstrated greater tumor growth inhibition effect in vivo without unexpected side effects, compared to PTX solution and PTX plus ATRA solution. These results suggest that PTX-loaded LHR nanoparticles can be considered as promising targeted delivery system for combination cancer chemotherapy to improve therapeutic efficacy and minimize adverse effects., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Alitretinoin is an endogenous retinoid related to vitamin A. Studies have shown that oral alitretinoin is effective and well tolerated in the treatment of severe chronic hand eczema. This review summarizes the clinical pharmacokinetic and pharmacodynamic data from a number of studies involving alitretinoin. These include the effect of food on the pharmacokinetics of alitretinoin, interactions between alitretinoin and ketoconazole, simvastatin or cyclosporin A, the effect of alitretinoin on the pharmacokinetics of a combined oral contraceptive, alitretinoin in seminal fluid after repeated dosing, and the pharmacokinetics of alitretinoin and its metabolites in a clinical setting.

Purpose: To develop a simple pharmacodynamic (PD) assay for the evaluation of the bioequivalence of topically applied retinoid products., Methods: Daily applications of products containing tretinoin or adapalene were made to the forearms of human subjects for up to 21 days. Percutaneous absorption was enhanced through the use of polyethylene film occlusion (5 h). Pharmacologic activity was assessed through the daily measurement of three cutaneous responses intimately linked to retinoid-induced changes in epidermal differentiation: (1) erythema; (2) exfoliation (scaling/peeling), and (3) increased transepidermal water loss., Results: The PD model exhibited the sensitivity and specificity required to function as a bioequivalence surrogate. It was possible to differentiate between: (1) three concentrations of tretinoin in a commercial cream product line; (2) two concentrations of tretinoin in a commercial gel product line; (3) different vehicles (gel vs. cream) containing the same concentration of tretinoin, and (4) tretinoin and adapalene at the same concentration. The applicability of this model for bioequivalence testing was established by showing that it had sufficient power to determine that three test tretinoin cream products and two approved generic tretinoin gel products were equivalent to their corresponding reference products., Conclusions: A surrogate PD model to assess retinoid bioequivalence has been developed., (Copyright © 2012 S. Karger AG, Basel.)

The active metabolite of vitamin A, retinoic acid (RA), is a powerful regulator of gene transcription. RA is also a therapeutic drug. The oxidative metabolism of RA by certain members of the cytochrome P450 (CYP) superfamily helps to maintain tissue RA concentrations within appropriate bounds. The CYP26 family--CYP26A1, CYP26B1, and CYP26C1--is distinguished by being both regulated by and active toward all-trans-RA (at-RA) while being expressed in different tissue-specific patterns. The CYP26A1 gene is regulated by multiple RA response elements. CYP26A1 is essential for embryonic development, whereas CYP26B1 is essential for postnatal survival as well as germ cell development. Enzyme kinetic studies have demonstrated that several CYP proteins are capable of metabolizing at-RA; however, it is likely that CYP26A1 plays a major role in RA clearance. Thus, pharmacological approaches to limiting the activity of CYP26 enzymes may extend the half-life of RA and could be useful clinically in the future.

The ability of a recently developed novel class of liposomes to promote dermal delivery of tretinoin (TRA) was evaluated. New penetration enhancer-containing vesicles (PEVs) were prepared adding to conventional phosphatidylcholine vesicles (control liposomes) different hydrophilic penetration enhancers: Oramix NS10 (OrNS10), Labrasol (Lab), Transcutol P (Trc), and propylene glycol (PG). Vesicles were characterized by morphology, size distribution, zeta potential, incorporation efficiency, stability, rheological behaviour, and deformability. Small, negatively charged, non-deformable, multilamellar vesicles were obtained. Rheological studies showed that PEVs had fluidity higher than conventional liposomes. The influence of the obtained PEVs on (trans)dermal delivery of tretinoin was studied by ex vivo diffusion experiments through new born pig skin using formulations having the drug both inside and outside the vesicles, having TRA only inside, in comparison with non-incorporated drug dispersions of the same composition used to produce the studied vesicles. Main result of these experiments was an improved cutaneous drug accumulation and a reduced transdermal TRA delivery (except for PG-PEVs). TRA deposition provided by PEVs was higher for dialysed than for non-dialysed vesicles. Further, the accumulation increased in the order: control liposomes

Background: Alitretinoin, like all retinoids, is teratogenic, and can only be given to women of childbearing potential if pregnancy is excluded and a strict contraceptive programme is followed., Aim: This study was designed to determine whether alitretinoin in the semen of men treated with alitretinoin poses a teratogenic risk to their female partners., Methods: In total, 24 healthy men aged 18-45 years received alitretinoin 20 mg (n = 12) or 40 mg (n = 12), once daily for 14 days. Subjects in the 40 mg dose group provided ejaculate at baseline, on day 1, before and approximately 4 h after dosing on day 2, and at follow-up on study day 21 (± 2)., Results: Alitretinoin and 4-oxo-alitretinoin were detected in 11 of the 12 semen samples. The highest level of alitretinoin in semen was 7.92 ng/mL. Assuming an ejaculate volume of 10 mL, the amount of drug transferred in semen would be about 80 ng, 1/375,000 of a single 30 mg capsule. Complete absorption of 80 ng of alitretinoin from semen, presuming a volume of distribution confined to 5 L of circulating blood in the partner, would lead to an increase in plasma alitretinoin concentration of 0.016 ng/mL, which appears to be negligible compared with measured endogenous plasma levels. Increases in plasma levels of related retinoids are also negligible., Conclusions: Alitretinoin in the semen of men receiving up to 40 mg of oral alitretinoin per day is unlikely to be associated with teratogenic risk in their female partners. Barrier contraception is therefore not required for men taking alitretinoin., (© 2011 The Author(s). Clinical and Experimental Dermatology © 2011 British Association of Dermatologists.)

Systemic therapies with retinoic acid (RA) can result in toxic side effects without yielding biologically effective levels in target tissues such as lung. The authors adapted a PARI LC Star nebulizer to create a tubular system for short-term inhalation treatment of guinea pigs using a water-miscible formulation of all-trans RA (ATRA) or vehicle. Based on the initial average weight, animals received an estimated average ATRA doses of either 0.32 mg·kg(-1) (low dose, 1.4 mM), or 0.62 mg·kg(-1) (medium dose, 2.8 mM), or 1.26 mg·kg(-1) (high dose, 5.6 mM) 20 minutes per day for 6 consecutive days. This system led to a rise of ATRA levels in lung, but not liver or plasma. Cellular lung levels of retinol, retinyl palmitate, and retinyl stearate also appeared to be unaffected (245.6 ± 10.7, 47.4 ± 3.4, and 132.8 ± 7.7 ng·g(-1) wet weight, respectively). The application of this aerosolized ATRA also induced a dose-dependent protein expression of the cellular retinol-binding protein 1 (CRBP-1) in lung, without apparent harmful side effects.

All-trans retinoic acid (RA) is a critical signaling molecule and its concentration is tightly regulated. Several P450 enzymes including CYP26A1, CYP2C8, and CYP3A4 have been proposed to be responsible for RA clearance in the liver but their quantitative importance has not been demonstrated. To determine the contribution of CYP26A1 to hepatic clearance of RA, CYP26A1 protein was quantified in 37 human liver microsomes (HLMs). CYP26A1 expression ranged from not detectable to 2.80pmol/mg microsomal protein. RA clearance by P450 enzymes abundant in human liver was measured in Supersomes. CYP2C8, CYP3A4, CYP3A5 and CYP3A7 metabolized RA with unbound K(m) values of 3.4-7.2microM and V(max) values of 2.3-4.9pmol/min/pmol P450, but were less efficient than CYP26A1 in clearing RA. Simulations performed for livers with varying P450 expression levels over a range of RA concentrations demonstrated that at both endogenous and therapeutic concentrations of RA, CYP26A1 is the primary enzyme responsible for 4-OH RA formation clearance. HLM incubation data showed that 4-OH RA formation velocity varied from 0.2 to 15.3pmol/min/mg microsomal protein and velocity in HLMs was significantly correlated (p<0.01) to CYP26A1, CYP3A4, and CYP3A5 protein content, but not to CYP2C8. When experimental data were scaled to in vivo clearances, the predicted hepatic clearance of RA (0.07L/min using combined Supersome data) was similar to the published in vivo clearance of RA. These findings suggest that CYP26A1 is the P450 isoform that should be targeted when designing RA metabolism blocking agents., (Copyright 2010 Elsevier Inc. All rights reserved.)

13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.

We tested the effects of poloxamer 188 on the preparation and characteristics of poly(lactice-co-glycolide) (PLGA) nanoparticles containing all-trans retinoic acid (ATRA). Spherical nanoparticles incorporated ATRA were prepared by an emulsification-diffusion method increasing concentrations of poloxamer 188 decreased nanoparticle size. The endothermic peak of ATRA at 183 degrees C disappeared in the nanoparticles and X-ray diffraction measurements showed the disappearance of characteristics of ATRA suggesting the change of the crystallinity of ATRA in the nanoparticles to an amorphous state. ATRA release in vitro increased as the concentration of poloxamer increased. The mean residence time of ATRA-loaded nanoparticles after the administration of a single intravenous dose to rats was longer than that of the sodium ATRA solution. The anti-cancer activity of ATRA-loaded nanoparticles in the human leukemia-60 cell line was similar to free ATRA. Thus, PLGA/poloxamer nanoparticles can provide a sustained release preparation of ATRA.

For a new anticancer drug carrier, we synthesized 4 compositions of amphiphilic stearic acidconjugated pullulan (SAP) and characterized them with FT-IR spectroscopy. Crystalline changes were verified by x-ray diffraction patterns before and after synthesis of the SAP conjugate. SAP nanoparticles were prepared by a diafiltration method, and the fluorescence spectroscopy using pyrene showed particle self-assembly in water. SAP nanoparticles were spherical in TEM photos, and particle size ranged between 200 approximately 500 nm in photon correlation spectroscopy. Release of all-trans-retinoic acid from the SAP nanoparticles was maintained over 5 weeks. For further study in vivo, we tested the cytotoxicity of SAP nanoparticles using an MTT assay, and cytotoxicity was augmented as the molar mass of stearic acid increased in human liver carcinoma HepG2 cells. Therefore, SAP nanoparticles might be a promising longterm delivery carrier for hydrophobic therapeutic molecules with the appropriate composition.

Retinoic acid derivatives have shown their greatest benefit in acute promyelocytic leukemia, but have also demonstrated pre-clinical anti-cancer effects in some solid tumors. Histone deacetylase inhibitors, by upregulating gene expression, are able to limit cancer cell proliferation and induce apoptosis. The combination of all-trans retinoic acid (ATRA) and the histone deacetylase inhibitor valproic acid has been previously studied in hematologic malignancies. We conducted a phase I two-step dose escalation trial of the liposomal ATRA analog ATRA-IV and divalproex sodium (Depakote) in nine patients with advanced solid tumors refractory to prior therapy. Side effects attributed to therapy had a severity

Background: Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis., Methods: RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography., Results: RALDH3 oxidized all-trans retinal with high catalytic efficiency (Vmax/Km=77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (Vmax/Km=27.4), and oxidized 13-cis retinal with lower catalytic efficiency (Vmax/Km=8.24). beta-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl2 activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity., Conclusions: These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates., General Significance: The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.

Background: Isotretinoin is effective in the treatment of severe acne and rosacea. Both parent drug and its main metabolite 4-oxo-isotretinoin are potentially teratogenic compounds and contain a carboxylic acid moiety. In the presence of ethanol, naturally occurring as well as synthetic retinoids also containing a carboxylic acid moiety are capable of undergoing an ethyl esterification with the metabolic formation of more lipophilic compounds with a much longer terminal half-life., Objectives: To determine if isotretinoin (13-cis-RA), its main metabolite 4-oxo-isotretinoin (4-oxo-13-cis-RA), and other possible metabolites in the presence or absence of ethanol are converted to their corresponding ethyl derivatives in patients with severe acne or rosacea after multiple isotretinoin dosing. In addition, pharmacokinetic parameters of the parent drug and its 4-oxo metabolite were determined., Patients/methods: Eleven patients with severe acne or rosacea were treated with isotretinoin daily for 3 months and investigated pharmacokinetically during 24 h after 1 month of treatment and for up to 28 days after discontinuation of therapy. A possible influence of ethanol was evaluated using a simple self-administered questionnaire and by measuring serum ethanol levels during treatment. The concentrations of isotretinoin, 4-oxo-isotretinoin and possible ethylated and nonethylated metabolites were measured by reverse-phase high-performance liquid chromatography., Results: Although seven of 11 patients had a considerable weekly alcohol intake, no endogenous synthesis of ethyl derivatives of isotretinoin, the main 4-oxo metabolite or the all-trans compounds was chromatographically detectable in any of the patients' plasma samples during the treatment period. Multiple dose pharmacokinetic data for the parent drug and its main metabolite were comparable to previous studies., Conclusions: The metabolism and pharmacokinetics of isotretinoin and its main metabolites are not influenced by ethanol during long-term isotretinoin treatment. After ceasing long-term isotretinoin therapy the recommended period of 1 month for using anticonceptive measures in fertile women seems adequate.

Alitretinoin is an endogenous retinoid and acts as a pan-agonist at retinoid receptors, binding with high affinity to both retinoic acid receptors and retinoid X receptors (RXR). Oral alitretinoin once daily is approved for use in patients with severe chronic hand eczema unresponsive to treatment with potent topical corticosteroids. In a large (n = 1032), randomized, double-blind, placebo-controlled, multicentre study (BACH) of up to 24 weeks' duration in adults with severe chronic hand eczema, significantly more patients in the alitretinoin 10 or 30 mg/day groups than in the placebo group responded to treatment with clear/almost clear hands, as assessed by the Physician Global Assessment (PGA) [primary endpoint]. In an extension phase of the BACH study, alitretinoin was effective in patients who relapsed after responding to initial treatment with the drug. Of patients who had responded to initial treatment with alitretinoin 30 mg/day, significantly more alitretinoin 30 mg/day than placebo recipients responded on the PGA with clear/almost clear hands during the extension phase (primary endpoint; 80% vs 8%). Of those who had responded to initial treatment with alitretinoin 10 mg/day, 48% of alitretinoin 10 mg/day and 10% of placebo recipients responded during the extension phase. Alitretinoin was generally well tolerated in clinical trials excluding pregnant women. The most common treatment-emergent adverse events and abnormal laboratory test results were consistent with those previously observed with other oral retinoids and RXR agonists.

Chitosan treated alginate microparticles were prepared with the purpose of incorporating all-trans retinoic acid (ATRA) using an inexpensive, simple and fast method, enhancing dermal localization and sustaining the release of ATRA into the skin. Microparticles characterization, drug-polymer interaction, release profile and in vitro skin retention were investigated. Microparticles presented spherical shape and drug loading capacity of 47%. The drug content of these microparticles was affected by ATRA concentration and by the solvent used and it was more weakly affected by chitosan concentration. The release of ATRA was also affected by chitosan concentration. Microparticles prepared with 0.4% chitosan (w/w) resulted in drug release with a more sustained profile. The results of in vitro retention studies showed that chitosan treated alginate microparticles decreased the drug retention in the stratum corneum (SC), where occur the skin irritation, but maintained the ATRA concentration in the deeper skin layers, where occur the pathologies treated with ATRA. Then, the microparticles developed in this work can be a good candidate to improve the topical therapy with retinoid.

Although all-trans retinoic acid (ATRA) is widely used in acute promyelocytic leukemia (APL), there is little data as to whether or not ATRA is useful for patients with liver and renal failure. A 63-year-old APL patient, complicated by Child-Pugh class A liver cirrhosis and chronic renal failure (creatinine 3.2 mg/dL), was successfully treated with 45 mg/m(2)/day of ATRA. With three courses of chemotherapy, complete remission has been maintained for four years in this patient. Serum trough and maximum ATRA concentration, and the area under the curve (AUC) were not elevated. These observations suggest that full-dose ATRA therapy might be safely applicable to such a complicated case with APL.

Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.

A highly sensitive, rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of retinamido-ester in rat plasma was developed and validated. A simplified protein precipitation with acetonitrile was employed for the sample preparation. The separation was carried out on an Agilent TC C18 column (150 mm x 4.6 mm ID, 5 microm particle size) with the mobile phase consisted of methanol-water-formic acid (93: 7: 0.1). Simvastatin was used as internal standard. The detection was performed on a trap-quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) scan mode via atmospheric pressure chemical ionization (APCI). The range of calibration curve was 0.05-50 ng x mL(-1) and the limit of quantification was 10 pg x mL(-1). The intra- and inter-day precision values were between 95.97% and 104.43%, and RSD was between 4.63% and 10.69%, respectively. This method was applied to determine the pharmacokinetic parameters. The main pharmacokinetic parameters of retinamido-ester after oral administration via gastric gavage of 2.5, 5, 10 mg x kg(-1) were as follows, T(1/2): (11.28 +/- 7.23), (8.90 +/- 3.82), (8.01 +/- 5.65) h; AUC(0-infinity): (103.41 +/- 61.46), (190.23 +/- 74.99), (421.66 +/- 229.20) ng x h x mL(-1); MRT: (6.31 +/- 0.75), (5.98 +/- 0.71), (6.18 +/- 0.97) h; CL/F: (30.10 +/- 13.67), (29.58 +/- 10.59), (31.18 +/- 17.51) L x h(-1) x kg(-1); Vd/F: (414.94 +/- 159.82), (356.16 +/- 139.85), (369.28 +/- 322.72) L x kg(-1), respectively.

Fast and sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method for the specific determination of viaminate in 14 kinds of rat tissues pre-treated with simple procedure was developed and validated. Biological samples were prepared by direct precipitation to skin, stomach, intestine, and liver and extracted by liquid-liquid extraction to lung, kidney, muscle, spleen, brain, fat, testes (male and female), eye, and heart. After addition of menaquinon as internal standard to tissue homogenate, the supernatant was injected into the isocratic chromatographic system using a Waters Symmetry C8 column and methanol-water-formic acid (93:7:0.1) as the eluent. The eluate was completely led into an APCI interface with selected ion monitoring mode and the analytes were quantified using triple quadrupole MS. The assays were successfully validated in the ranges 0.02-20 ng/mL for lung, 0.02-10 ng/mL for kidney, spleen, muscle, brain, fat, eye, and heart, 0.05-10 ng/mL for testicle, 0.4-100 ng/mL for liver, skin and intestine, and 1.0-200 ng/mL for stomach. The accurate and precise studies showed good reproducibility with coefficients of variation below 8.5% and the recoveries range from 90 to 109%. The analytes were chemically stable under all relevant conditions and the assays were applied in tissue distribution study. The results showed that the viaminate concentration was high in skin, low in kidney, and almost undetectable in eye and brain.

The pharmacokinetics of all-trans-retinoic acid (ATRA), an anti-cancer drug was highly variable due to its poor aqueous solubility. In this study, we investigated the pharmacokinetics of ATRA in male Wistar rats following intravenous administration of the ATRA loaded tributyrin emulsion. In vitro, the ATRA emulsion was proved binding to apolipoprotein(s). In vivo, the clearance of ATRA was significantly reduced by formulating into the tributyrin emulsion, leading to higher AUCs. Co-administration with 17alpha-ethynylestradiol, a compound known to upregulate the activity of low-density lipoprotein receptors in tissues, significantly increased the K(e), V, and CL of ATRA. The variation of plasma AUCs after administering the ATRA emulsion to the healthy rats was two times less than that after the ATRA solution. The IC(50) in ATRA of the ATRA emulsion for the Caco-2 carcinoma cells was 3.8 microg/mL lower than 6 microg/mL of the ATRA solution. The IC(50) of the emulsion for the HepG2 carcinoma cells was 2.8 microg/mL, while IC(50) was not achieved with the ATRA solution over the test concentration range. The finding indicated that the tributyrin emulsion could be used as a carrier for ATRA and enhances the drug effect by reducing the clearance and increasing the in vitro activity.

Active derivatives of vitamin A are essential in physiological processes such as cell growth, differentiation, morphogenesis and development. The biological functions of vitamin A are mediated through the retinoid acid receptors (RARs) and retinoid X receptors (RXRs). Aryl hydrocarbon receptor (AhR) agonists such as planar halogenated compounds are known to interfere with vitamin A homeostasis in both field and laboratory studies. In this study, we have investigated the molecular interactions between vitamin A and AhR signalling pathways using juvenile Atlantic salmon and agonists for both receptor pathways. Groups of juvenile salmon were treated with all-trans- and 9-cis-retinoic acid mixture (7:3 ratio) dissolved in DMSO (dimethyl sulfoxide) at 0.1, 1 and 10 mg/kg fish weight. The mixture was force fed singly or in combination with 0.1 mg 3,3',4,4'-tetrachlorobiphenyl (co-planar congener 77)/kg fish weight dissolved in DMSO. Liver samples were collected 3 days after PCB-77 exposure. A separate group exposed to combined retinoic acid (1 mg/kg for 5 days) and PCB-77, was sampled at 3, 7 and 14 days after PCB-77 exposure. Liver samples collected from all exposure groups were analyzed for gene (RARalpha, AhR2alpha, AhR2beta, CYP1A1, UGT1 and GSTpi) expression using real-time PCR and activity (7-ethoxyresorufin O-deethylase (EROD), UGT and GST) using biochemical methods with specific substrates. Our data showed that exposure to RA alone did not produce a significant increase of RARalpha mRNA levels, and the presence of PCB-77 attenuated the expression of RARalpha in RA dose- and time-specific manner. In addition, RA produced a dose-dependent increase of CYP1A1 mRNA and activity (EROD) levels without concomitant increase in AhR2 isoforms. When administered alone, PCB-77 produced increased CYP1A1, UGT1 and GSTpi mRNA and enzyme levels. The PCB-77-induced CYP1A1, UGT1 and GSTpi (mRNA and activity) levels were modulated by RA, in a parameter and dose-specific manner. In general, our data show an interaction between vitamin A and AhR signalling that may affect retinoid homeostasis in fish.

Folic acid, conjugated to poly(ethylene glycol)-distearoylphosphatidylethanolamine (folate-PEG-DSPE), was used to target emulsions of all-trans retinoic acid (ATRA) to folate receptor-overexpressing tumor cells. Two kinds of ATRA-incorporated folate-tethered emulsions (ATRA-FTE 2000/3400) were prepared using soybean oil, egg phosphatidylcholine and folate-PEG-DSPE with different PEG length. As a control, ATRA-incorporated non-tethered emulsion (ATRA-NTE) was prepared by using PEG2000-DSPE without folate instead of using folate-PEG-DSPE. The mean particle diameters of ATRA-FTE 2000/3400 were about 100-130 nm. The cellular uptake in KB cells of fluorescence-labeled ATRA-FTE 3400 was determined with HPLC (for ATRA) and confocal microscopy (for lipid emulsion). The growth inhibitory activity of ATRA was evaluated by MTT assay. The folate ligands in emulsion increased the cellular uptake of ATRA about 3-fold and 1.6-fold in ATRA-FTE 3400 and ATRA-FTE 2000, respectively. Growth inhibitory activity of ATRA-FTE 3400 in KB cells was higher than that of ATRA-NTE at the same dose. Whereas the growth inhibitory effect in MCF-7 cells of ATRA was similar between ATRA-NTE and ATRA-FTE 3400. The addition of free folate significantly reduced the uptake of ATRA regardless of the length of PEG attached to folate. Folate-tethered lipid emulsion showed effective and selective delivery to the folate receptor-abundant carcinomas, suggesting a potential for targeted delivery of anticancer agents.

Purpose: All-trans retinoic acid (ATRA) polymeric micelles were developed for parenteral administration. The distribution characteristics and antitumor activities of ATRA polymeric micelles were evaluated after intravenous administration to mice bearing CT26 solid tumors., Methods: ATRA incorporated in poly(ethylene glycol)-poly(benzyl aspartate) block copolymer was prepared by the evaporation method. The levels of [3H]ATRA in blood and tissue including tumor were determined by measuring the radioactivity after injection into mice. The tumor volume and the survival of the mice were determined to assess the anticancer activity., Results: The delivery of ATRA by polymeric micelles prolonged the blood circulation and enhanced the accumulation of ATRA in the tumor tissue compared with the administration of free ATRA. Tumor growth was significantly delayed and the survival time of mice was prolonged following the treatment by ATRA polymeric micelles demonstrating the improved anticancer activity of ATRA., Conclusion: Polymeric micelles are a promising and effective carrier of ATRA in order to enhance tumor delivery and they have a promising potential application in the treatment of solid tumors.

In emphysema, the lung cannot spontaneously regenerate lost alveolar tissue. Treatment with retinoic acid (RA) in rodent models of emphysema induces alveolar regeneration. However, some animal studies have failed to show regeneration when using different species and strains. We have previously shown that dexamethasone (Dex) treatment of newborn TO outbred strain mice permanently disrupts alveolar development. Later RA treatment restores alveolar architecture to normal. To determine whether this model of alveolar regeneration is strain specific, our protocol was repeated with two new outbred mouse strains. ICR and NIHS mice received Dex from Postnatal Days 4 to P15 (P4- P15). From P46 to P57, mice received RA (2 mg/kg) or vehicle. An additional ICR group received 5x RA (10 mg/kg) from P46 to P57. Control groups received vehicle at both treatment points. All mice were killed at P90 and lung morphology analyzed. Dex-treated ICR and NIHS mice showed increased mean alveolar chord length (Lm) and reduced alveolar surface area (SA) and SA/lung volume (SA/LV) compared with controls. RA-treated NIHS mice showed return of Lm, SA, and SA/LV toward control values, indicating alveolar regeneration. ICR RA group mice did not regenerate, but 5x RA mice showed Lm, SA, and SA/LV values consistent with alveolar regeneration. In conclusion, the Dex-treated mouse model of emphysema is robust and repeatable in different strains. RA-induced alveolar regeneration is not a strain-specific phenomenon. RA dose threshold for inducing alveolar regeneration is higher in ICR mice, suggesting a difference in retinoid pharmacokinetics between strains. These results provide a possible explanation for previous failed studies of RA-induced alveolar regeneration.

Retinoids promote lung alveolarization in animal models and were administered to patients as part of the Feasibility of Retinoid Therapy for Emphysema (FORTE) study. This FORTE substudy investigated the pharmacokinetic profiles of 2 retinoic acid isomers-all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid (13-cRA)-in subjects with emphysema, evaluated strategies to overcome self-induced ATRA catabolism, and identified pharmacodynamic relationships. Comprehensive and limited pharmacokinetics were obtained at multiple visits in emphysema subjects treated with placebo (n = 30), intermittent dosing (4 days/week) with low-dose ATRA (1 mg/kg/day, n = 21), or high-dose ATRA (2 mg/kg/day, n = 25) or daily administration of 13-cRA (1 mg/kg/day, n = 40). High-dose ATRA produced the highest peak plasma ATRA Cmax. However, at follow-up, plasma ATRA C(max) was significantly decreased from baseline in subjects whose day 1 levels exceeded 100 ng/mL (P < .0001). In contrast, administration of 13-cRA produced lower plasma ATRA C(max) (<100 ng/mL), but the levels were significantly higher at follow-up than those on day 1 (P < .001). Plasma ATRA levels as determined on day 1 correlated with changes in pulmonary diffusing capacity at 6 months, consistent with concentration-dependent biologic effects (r2 = -0.25). The authors conclude that intermittent therapy with high-dose ATRA produced the greatest ATRA exposure, but alternative approaches for limiting self-induced ATRA catabolism should be sought.

All trans retinoic acid (ATRA) combined with chemotherapy has become the mainstay of treatment for patients with acute promyelocytic leukemia (APL). Renal dysfunction (RD) is commonly seen in patients with APL. We describe a patient with APL and multi-organ failure, who was on chronic veno-venous hemofiltration followed by hemodialysis (HD) and later peritoneal dialysis (PD), who received ATRA. ATRA levels were assessed as the body clearance of ATRA in children on HD and/or PD was unknown. Neither HD nor PD significantly affected ATRA levels, suggesting that dose modifications of ATRA may not be necessary for children with these forms of renal replacement therapy., (2007 Wiley-Liss, Inc)

Purpose: Novel potent C-4 azolyl retinoic acid metabolism blocking agents (RAMBAs)-VN/14-1, VN/50-1, VN/66-1, VN/67-1, and VN/69-1, have been synthesized and investigated for their in vitro and in vivo effects against breast and prostate cancers. These RAMBAs, in addition to being potent inhibitors of all-trans-retinoic acid (ATRA) metabolism have potent anti-cancer properties and in vivo anti-tumor efficacies as characterized in breast and prostate cancer models. Here we determined the toxicity and pharmacokinetics (PK) of these various RAMBAs., Methods: Preliminary acute toxicity studies of these RAMBAs were carried out using Swiss NIH mice. The toxicity profile of the RAMBAs was evaluated relative to ATRA. Three different doses (8.3, 33, and 100 micromol/kg/day) of ATRA and RAMBAs were administered on a daily basis subcutaneously for 14 days to the mice. Clinical signs of toxicity alopecia, scaly skin, and loss of body weight in the mice were observed during the study and the maximum tolerated dose was determined. PK of selected agents (VN/14-1, VN/50-1, and VN/66-1) was studied in Balb/C mice after a single dose subcutaneous administration. Plasma concentrations of the agents were quantitatively determined using a high-performance liquid chromatographic method with ultraviolet detection. Plasma concentration versus time profiles were fit to various PK structural models and relevant PK parameters were estimated., Results: VN/66-1 and VN/69-1 were found to be the least toxic even at the highest doses when compared to the other RAMBAs and ATRA. VN/66-1 had the longest half-life, the slowest clearance, and the greatest exposure., Conclusions: Based on PK characteristics and toxicity studies, VN/66-1 appeared to be the most favorable agent. However, both VN/14-1 and VN/66-1 are our leads based on the fact that VN/14-1 has been found to be highly effective in endocrine-sensitive and -resistant breast cancer cells and tumors with little toxicity. Our findings provide valuable information that will be used to select RAMBAs and establish therapeutic regimens that provide optimal efficacy with minimal toxicity.

To evaluate the feasibility, efficacy, and biologic effects of weekly liposome-encapsulated all-trans retinoic acid (ATRA-IV) plus interferon alpha2b (IFN) in patients with advanced renal cell carcinoma (RCC). Twenty-six patients with metastatic RCC were treated on a phase 1/2 trial with weekly ATRA-IV and IFN SQ daily 5 d/wk. Twelve patients received ATRA-IV at three dose levels (60, 75, and 90 mg/m2) according to phase 1 methodology, and 14 additional patients received 90 mg/m2. Response was assessed according to an intention-to-treat analysis. Serum retinoic acid (RA) concentrations were assayed and peripheral blood mononuclear cell mRNA expression of RA and IFN-inducible genes (RARalpha, RARbeta2, IRF1, CRABP2, and TRAIL) were examined. No dose limiting toxicities occurred at 60 mg/m2; grade 3 leukopenia affected 1/6 patients at 75 mg/m2, whereas 3 patients received 90 mg/m2 without a dose limiting toxicities. Fourteen additional patients received 90 mg/m2 ATRA-IV without grade 3/4 toxicity. Five of 26 (19%) patients achieved a major response, with a median duration of 14 months (range 9 to 23); 9 additional patients (41%) demonstrated stable disease or minor response lasting > or =4 months. No significant differences in serum (RA) after ATRA infusion were detected between weeks 1 and 8 of treatment. Peripheral blood mononuclear cell mRNA expression did not correlate with clinical response. The addition of weekly ATRA-IV to IFN therapy is feasible and well tolerated, resulting in sustainable increased serum (RA). This regimen demonstrates antitumor activity in metastatic RCC, and suggests ATRA-IV augments IFN therapy.

Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg(-1)) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA.

Despite its known biological effect on epithelial cells, 13- CIS-retinoic acid shows low binding affinity to either cellular retinoic acid-binding proteins or nuclear retinoid receptors compared to its isomer all- TRANS-retinoic acid. We have postulated a prodrug-drug relation with 13- CIS-retinoic acid which isomerizes to all- TRANS-retinoic acid. On the other hand, the biological effects of these two compounds can differ in the widely used cell culture models of HaCaT and normal primary keratinocytes. In this study, we seeded HaCaT and normal keratinocytes at high densities leading to early confluence in order to imitate high keratinocyte proliferation, such as in acne and psoriasis, while to model decreased keratinocyte proliferation, as in aged and steroid-damaged skin, cells were seeded at a low density. High performance liquid chromatography was administered to examine retinoid uptake and metabolism in monolayer HaCaT and normal keratinocyte cultures and the 4-methylumbelliferyl heptanoate assay to estimate cell growth at different cell densities. Major qualitative and quantitative differences were detected in the two cell types regarding intracellular 13- CIS-retinoic acid isomerization to all- TRANS-retinoic acid. On the other hand, the two retinoic acid isomers showed similar effects on cell growth of both cell types tested with increasing proliferation at low cell densities, but being rather inactive at high ones in normal keratinocytes and exhibiting an antiproliferative effect in HaCaT keratinocytes. The missing effect of retinoids on cell proliferation in high seeding densities of normal keratinocytes may indicate that the normalizing activity of retinoids on hyperkeratotic diseases, such as acne or psoriasis, is likely to be carried out by modulation of cell differentiation than cell growth. On the other hand, induced keratinocyte proliferation in low seeding densities may provide an explanation for the acanthosis induced by topical retinoids in aged and steroid-damaged skin.

The anticarcinogenic activities of retinoic acid (RA) are believed to be mediated by the nuclear RA receptor (RAR) and by the RA-binding protein cellular RA-binding protein-II (CRABP-II). In MCF-7 mammary carcinoma cells, growth inhibition by RA entails an early cell cycle arrest followed by induction of apoptosis. Here, we aimed to obtain insights into the initial cell cycle response. We show that a 3- to 5-h RA pulse is sufficient for inducing a robust growth arrest 2 to 4 days later, demonstrating inhibition of the G1-S transition by RA is triggered by immediate-early RAR targets and does not require the continuous presence of the hormone throughout the arrest program. Expression array analyses revealed that RA induces the expression of several genes involved in cell cycle regulation, including the p53-controlled antiproliferative gene B-cell translocation gene, member 2 (Btg2) and the BTG family member Tob1. We show that induction of Btg2 by RA does not require de novo protein synthesis and is augmented by overexpression of CRABP-II. Additionally, we identify a RA response element in the Btg2 promoter and show that the element binds retinoid X receptor/RAR heterodimers in vitro, is occupied by the heterodimers in cells, and can drive RA-induced activation of a reporter gene. Hence, Btg2 is a novel direct target for RA signaling. In concert with the reports that Btg2 inhibits cell cycle progression by down-regulating cyclin D1, induction of Btg2 by RA was accompanied by a marked decrease in cyclin D1 expression. The observations thus show that the antiproliferative activity of RA in MCF-7 cells is mediated, at least in part, by Btg2.

The epithelial barrier is determined primarily by intercellular tight junctions (TJs). We have demonstrated previously that all-trans retinoic acid (atRA) plays an important role in forming functional TJs through a specific retinoic acid receptor (RAR)/retinoid X receptor heterodimer in epithelial cells. However, the physiological relevance of retinoic acids (RAs) in maintaining the epithelial integrity remains to be examined. Here, we show that several types of RA, including atRA, promote the barrier function of epithelial TJs. Conversely, RA depletion in the cells by overexpressing CYP26s, cytochrome P450 enzymes specifically involved in the metabolic inactivation of RAs, induces an increase of permeability as measured by two differently sized tracer molecules, inulin and mannitol. This RA-mediated enhancement of barrier function is potentially associated with the increased expression of TJ-associated genes such as occludin, claudin-1, claudin-4, and zonula occludens-1. We also found that RARalpha is a preferential regulator of the epithelial barrier in vitro. Studies of murine experimental colitis, which is characterized by increased gut permeability, reveal that RARalpha stimulation significantly attenuates the loss of the epithelial barrier during colitis in vivo. Our results suggest that cellular RA bioavailability determines the epithelial integrity, because it is a critical regulator for barrier protection during mucosal injuries.

Abnormal dendritic cell differentiation and accumulation of immature myeloid suppressor cells (ImC) is one of the major mechanisms of tumor escape. We tested the possibility of pharmacologic regulation of myeloid cell differentiation using all-trans-retinoic acid (ATRA). Eighteen patients with metastatic renal cell carcinoma were treated with ATRA followed by s.c. interleukin 2 (IL-2). Eight healthy individuals comprised a control group. As expected, the cancer patients had substantially elevated levels of ImC. We observed that ATRA dramatically reduced the number of ImC. This effect was observed only in patients with high plasma concentration of ATRA (>150 ng/mL), but not in patients with lower ATRA concentrations (<135 ng/mL). Effects of ATRA on the proportions of different dendritic cell populations were minor. However, ATRA significantly improved myeloid/lymphoid dendritic cell ratio and the ability of patients' mononuclear cells to stimulate allogeneic T cells. This effect was associated with significant improvement of tetanus-toxoid-specific T-cell response. During the IL-2 treatment, the ATRA effect was completely eliminated. To assess the role of IL-2, specimens from 15 patients with metastatic renal cell carcinoma who had been treated with i.v. IL-2 alone were analyzed. In this group also, IL-2 significantly reduced the number and function of dendritic cells as well as T-cell function. These data indicate that ATRA at effective concentrations eliminated ImC, improved myeloid/lymphoid dendritic cell ratio, dendritic cell function, and antigen-specific T-cell response. ATRA treatment did not result in significant toxicity and it could be tested in therapeutic combination with cancer vaccines.