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This paper reports a smart intracellular nanocarrier for sustainable and controlled drug release in non-invasive neuroregeneration. The nanocarrier is composed by superparamagnetic iron oxide-gold (SPIO-Au) core-shell nanoparticles (NPs) conjugated with porous coordination cages (PCCs) through the thiol-containing molecules as bridges. The negatively charged PCC-2 and positively charged PCC-3 are compared for intracellular targeting. Both types result in intracellular targeting via direct penetration across cellular membranes. However, the pyrene (Py)-PEG-SH bridge enabled functionalization of SPIO-Au NPs with PCC-3 exhibits higher interaction with PC-12 neuron-like cells, compared with the rhodamine B (RhB)-PEG-SH bridge enabled case and the stand-alone SPIO-Au NPs. With neglectable toxicities to PC-12 cells, the proposed SPIO-Au-RhB(Py)-PCC-2(3) nanocarriers exhibit effective drug loading capacity of retinoic acid (RA) at 13.505 μg/mg of RA/NPs within 24 h. A controlled release of RA is achieved by using a low-intensity 525 nm LED light (100% compared to 40% for control group within 96 h)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Neurogenic niches constitute a powerful endogenous source of new neurons that can be used for brain repair strategies. Neuronal differentiation of these cells can be regulated by molecules such as retinoic acid (RA) or by mild levels of reactive oxygen species (ROS) that are also known to upregulate RA receptor alpha (RARα) levels. Data showed that neural stem cells from the subventricular zone (SVZ) exposed to blue light (405nm laser) transiently induced NADPH oxidase-dependent ROS, resulting in β-catenin activation and neuronal differentiation, and increased RARα levels. Additionally, the same blue light stimulation was capable of triggering the release of RA from light-responsive nanoparticles (LR-NP). The synergy between blue light and LR-NP led to amplified neurogenesis both in vitro and in vivo, while offering a temporal and spatial control of RA release. In conclusion, this combinatory treatment offers great advantages to potentiate neuronal differentiation, and provides an innovative and efficient application for brain regenerative therapies., Statement of Significance: Controlling the differentiation of stem cells would support the development of promising brain regenerative therapies. Blue light transiently increased reactive oxygen species, resulting in neuronal differentiation and increased retinoic acid receptor (RARα) levels. Additionally, the same blue light stimulation was capable of triggering the release of RA from light-responsive nanoparticles (LR-NP). The synergy between blue light and LR-NP led to amplified neurogenesis, while offering a temporal and spatial control of RA release. In this sense, our approach relying on the modulation of endogenous stem cells for the generation of new neurons may support the development of novel clinical therapies., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Pegylation method is widely used to prolong the blood circulation time of proteins and nanoparticles after intravenous administration, but the effect of surface poly (ethylene glycol) (PEG) chain length on oral absorption of the pegylated nanoparticles is poorly reported. The aim of our study was to investigate the influence of PEG corona chain length on membrane permeability and oral bioavailability of the amphiphilic pegylated prodrug-based nanomicelles, taking all trans-retinoic acid (ATRA) as a model drug. The amphiphilic ATRA-PEG conjugates were synthesized by esterification reaction between all trans-retinoic acid and mPEGs (mPEG500, mPEG1000, mPEG2000, and mPEG5000). The conjugates could self-assemble in aqueous medium to form nanomicelles by emulsion-solvent evaporation method. The resultant nanomicelles were in spherical shape with an average diameter of 13-20 nm. The drug loading efficiency of ATRA-PEG500, ATRA-PEG1000, ATRA-PEG2000, and ATRA-PEG5000 was about 38.4, 26.6, 13.1, and 5.68 wt%, respectively. With PEG chain length ranging from 500 to 5000, ATRA-PEG nanomicelles exhibited a bell shape of chemical stability in different pH buffers, intestinal homogenate and plasma. More importantly, they were all rapidly hydrolyzed into the parent drug in hepatic homogenate, with the half-time values being 0.3-0.4h. In comparison to ATRA solution and ATRA prodrug-based nanomicelles, ATRA-PEG1000 showed the highest intestinal permeability. After oral administration, ATRA-PEG2000 and ATRA-PEG5000 nanomicelles were not nearly absorbed, while the oral bioavailability of ATRA-PEG500 and ATRA-PEG1000 demonstrated about 1.2- and 2.0-fold higher than ATRA solution. Our results indicated that PEG1000 chain length of ATRA-PEG prodrug nanomicelles has the optimal oral bioavailability probably due to improved stability and balanced mucus penetration capability and cell binding, and that the PEG chain length on a surface of nanoparticles cannot exceed a key threshold with the purpose of enhancement in oral bioavailability., (Copyright © 2015. Published by Elsevier B.V.)

Background: Ambient particulate matters (PMs) are known as inducers that adversely affect a variety of human organs., Objectives: In this study, we aimed to evaluate the influence of PMs on the permeation of drugs and sunscreens via the skin. The role of skin-barrier properties such as the stratum corneum (SC) and tight junctions (TJs) during the delivery process was explored., Methods: This work was conducted using both in vitro and in vivo experiments in pigs to check the responses of the skin to PMs. PMs primarily containing heavy metals (1648a) and polycyclic aromatic hydrocarbons (PAHs, 1649b) were employed to treat the skin., Results: According to the transepidermal water loss (TEWL), 1649b but not 1648a significantly disrupted the SC integrity by 2-fold compared to the PBS control. The immunohistochemistry (IHC) of cytokeratin, filaggrin, and E-cadherin exhibited that 1649b mildly damaged TJs. The cytotoxicity of keratinocytes and skin fibroblasts caused by 1649b was stronger than that caused by 1648a. The 1649b elicited apoptosis via caspase-3 activation. The proteomic profiles showed that PMs upregulated Annexin A2 by >5-fold, which can be a biomarker of PM-induced barrier disruption. We found that the skin uptake of ascorbic acid, an extremely hydrophilic drug, was increased from 74 to 112 μg/g by 1649b treatment. The extremely lipophilic drug tretinoin also showed a 2.6-fold increase of skin accumulation. Oxybenzone and dextran absorption was not affected by PMs. The in vivo dye distribution visualized by fluorescence microscopy had indicated that 1649b intervention promoted permeant partitioning into SC., Conclusions: Caution should be taken in exposing the skin to airborne dust due to its ability to reduce barrier function and increase the risk of drug overabsorption, although this effect was not very marked., (Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Little is known about the contribution of different tissues to whole-body vitamin A (VA) kinetics in neonates. Here, we have used model-based compartmental analysis of tissue tracer kinetic data from unsupplemented (control) and VA-retinoic acid (VARA)-supplemented neonatal rats to determine VA kinetics in specific tissues under control and supplemented conditions. First, compartmental models for retinol kinetics were developed for individual tissues, and then an integrated compartmental model incorporating all tissues was developed for both groups. The models predicted that 52% of chylomicron (CM) retinyl ester was cleared by liver in control pups versus 22% in VARA-treated pups, whereas about 51% of VA was predicted to be extrahepatic in 4- to 6-day-old unsupplemented neonatal rats. VARA increased CM retinyl ester uptake by lung, carcass, and intestine; decreased the release into plasma of retinol that had been cleared by liver and lung as CM retinyl esters; stimulated the uptake of retinol from plasma holo-retinol binding protein into carcass; and decreased the retinol turnover out of the liver. Overall, neonatal VA trafficking differed from that previously described for adult animals, with a larger contribution of extrahepatic tissues to CM clearance, especially after VA supplementation, and a significant amount of VA distributed in extrahepatic tissues., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Vitamin A (VA) metabolism in neonates is virtually uncharacterized. Our objective was to develop a compartmental model of VA metabolism in unsupplemented and VA-supplemented neonatal rats. On postnatal day 4, pups (n = 3/time) received 11,12-[(3)H]retinol orally, in either oil (control) or VA combined with retinoic acid (VARA) [VA (∼6 mg/kg body weight) + 10% retinoic acid]. Plasma and tissues were collected at 14 time points up to 14 days after dose administration. VARA supplementation rapidly, but transiently, increased total retinol mass in plasma, liver, and lung. It decreased the peak fraction of the dose in plasma. A multi-compartmental model developed to fit plasma [(3)H]retinol data predicted more extensive recycling of retinol between plasma and tissues in neonates compared with that reported in adults (144 vs. 12-13 times). In VARA pups, the recycling number for retinol between plasma and tissues (100 times) and the time that retinol spent in plasma were both lower compared with controls; VARA also stimulated the uptake of plasma VA into extravascular tissues. A VARA perturbation model indicated that the effect of VARA in stimulating VA uptake into tissues in neonates is both dramatic and transient., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

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.)

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.

Vitamin A (VA) is stored in tissues predominantly as retinyl esters (REs), which provide substrate for the production of bioactive retinoids. Retinoic acid (RA), a principal metabolite, has been shown to induce postnatal lung development. To better understand lung RE storage, we compared VA (given as retinyl palmitate), RA, and a nutrient-metabolite combination, VARA, given orally on postnatal days 5-7, for their ability to increase lung RE in neonatal rats. VARA increased lung RE significantly [ approximately 14, 2.4, 2.1, and <1 nmol/g for VARA, VA, RA, and control (C), respectively; P < 0.001]; the increase by VARA was more than additive compared with the effects of VA and RA alone. Lung histology and morphometry were unchanged. In a 6 h metabolic study, providing [(3)H]retinol with VARA, compared with VA or C, increased the uptake of newly absorbed (3)H by 3-fold, indicating that VARA stimulated the uptake of [(3)H]retinol and its retention as [(3)H]RE in neonatal lungs. After cessation of VARA, lung RE remained increased for 9 d afterward, through the period of alveolar development. In conclusion, VARA, a 10:1 nutrient-metabolite combination, increased lung RE significantly compared with VA alone and could be a promising therapeutic option for enhancing the delivery of VA to the lungs.

A new strategy for the skin delivery of bioactive compounds has been developed, using enzymes involved in the maintenance of the epidermal barrier function and the enzymatic transformation of corresponding precursors. This new strategy has been tested with regard to two enzymatic activities of the skin barrier: extracellular glucosidase and esterase/lipase. An analysis of the requirements for the glycosidic bond hydrolysis of any glycoconjugate by beta-glucocerebrosidase indicates that the release of the moiety linked to the glucose unit is obtained as long as the glycosidic bond being broken is not hindered, and as long as the leaving group property of the released moiety is good enough. This strategy was first applied to the release of the antioxidant delta-tocopherol. It was then extended to retinoic acid by introducing a spacer between the glucose unit and the bioactive moiety. This spacer was either a good leaving group such as hydroquinone, or a structure akin to a ceramide, namely glycerol. In these conditions, beta-glucocerebrosidase releases the complex spacer-active compound that is cleaved by an esterase. One of the advantages of this strategy lies in the slow release of the bioactive compound, extending in time its effect and most likely its tolerance, as is the case for retinoic acid.

Nuclear receptors are ligand-modulated transcription factors regulated by interactions with corepressors and coactivators, whose functions are not fully understood. Acute promyelocytic leukemia (APL) is characterized by a translocation, t(15;17), that produces a PML/RARalpha fusion oncoprotein, whose abnormal transcriptional function is successfully targeted by pharmacologic levels of all-trans-retinoic acid (ATRA). Mutations in the ligand-binding domain of PML/RARalpha that confer resistance to ATRA have been studied by expression in nonhematopoietic cells, such as Cos-1. Here, we show that ATRA binding and transcriptional activation by the same PML/RARalpha mutant differ markedly between nonhematopoietic and leukemic cell lines. Differential expression of the corepressor isoform silencing mediator for retinoid and thyroid receptors beta (SMRTbeta) correlates with increased ligand binding and transcription by the mutant PML/RARalpha. Transient and stable overexpression of SMRTbeta in hematopoietic cells that only express SMRTalpha increased ATRA binding, ligand-induced transcription, and ATRA-induced cell differentiation. This effect may not be limited to abnormal nuclear receptors, because overexpression of SMRTbeta increased ATRA-induced binding and transcriptional activation of wild-type receptors PML/RARalpha and RARalpha. Our results suggest a novel role for the SMRTbeta isoform whereby its cell-specific expression may influence the binding and transcriptional capacities of nuclear receptors, thus providing new evidence of distinct functions of corepressor isoforms and adding complexity to transcriptional regulation.

A simple reversed phase high performance liquid chromatographic (HPLC) method was developed for the separation and quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA), and other retinoids in rat plasma. VN/14-1RA, alone or in combination with ATRA, is effective at inhibiting the proliferation of prostate and breast cancer cell lines in vitro. Aliquots of rat plasma were spiked with the retinoids followed by addition of acetonitrile for precipitation of plasma proteins. The decanted supernatant was evaporated under a stream of nitrogen and reconstituted in acetonitrile. Analysis was accomplished by injection of an aliquot of the reconstituted sample into an HPLC system consisting of a Zorbax Rx-C18 column and a diode array detector. A mobile phase composed of ammonium acetate (0.1 M), acetic acid solution (2% (v/v)) and methanol at a flow rate of 1.0 mL/min was used for gradient elution. The recoveries for all compounds ranged from 65 to 85% regardless of the concentrations examined. The HPLC assay was linear over the range 0.10-5.0 microg/mL (CV < 10%) with a limit of quantification of 100 ng/mL for VN/14-1RA. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration-time profile for VN/14-1RA after intravenous administration. The mean terminal elimination half-life (t(1/2)) was 19.0 +/- 3.2 min. This HPLC method is useful for the analysis and evaluation of the pharmacokinetics of VN/14-1RA in rats.

Diethyl dithiocarbamate (DEDTC) has been reported to be a more powerful inhibitor of all-trans-retinoic acid (ATRA) in vitro metabolism than the well-established cytochrome P450 (CYP) inhibitor ketoconazole (KC). In recent years grapefruit juice (GJ) has been shown to be able to increase the oral bioavailability of several drugs by inhibiting intestinal CYP. This study investigated the in vivo effect of these CYP inhibitors on the pharmacokinetics of ATRA. The latter was administered to rats as a constant-rate intravenous (i.v.) infusion (0.48 mg h(-1) kg(-1)) during 10 h and orally (1.6 mg kg(-1)). DEDTC (320 mg kg(-1) x 2 i.v., 6.4 and 32 mg kg(-1) per os (p.o.)) did not change the ATRA concentration-time profiles, whereas KC (320 and 32 mg kg(-1) p.o.)--with i.v. infused or orally dosed ATRA--increased the mean concentration-time curve value by 160% and 78%, respectively. A high dose of DEDTC (320 mg kg(-1) p.o.) caused a marked decrease in plasma levels of ATRA. GJ (6.4 ml kg(-1) p.o.) did not affect the plasma levels of ATRA. It is concluded that the in vivo effect of CYP inhibitors (DEDTC and KC) on the elimination rate of ATRA is qualitatively different from that expected from in vitro studies.

Retinoids are important modulators for cell growth and differentiation of normal skin. In plasma, retinol is transported coupled to plasma retinol-binding protein. In this study, we investigated gene and protein expression of RPE65, a putative receptor for plasma retinol-binding protein in human epidermal keratinocytes. We performed real-time PCR analysis to evaluate expression of RPE65 mRNA in proliferating and differentiating keratinocytes. Immunoblotting with anti-RPE65 antibody shows distinct reactivity to a 61-kDa protein. Indirect immunofluorescence on normal human epidermis reveals cell surface labeling of keratinocytes. Laser scan microscopy exhibits colocalization of plasma retinol-binding protein and RPE65 on cultured keratinocytes. Internalization experiments with [3H]retinoic acid-retinol-binding protein complex in the presence and absence of excess of retinol-binding protein indicates receptor-dependent uptake of retinoids. We further show isolation of RPE65 protein by affinity chromatography from lysates of keratinocytes using a retinol-binding protein-matrix gel column. In summary, we demonstrate mRNA and protein expression of RPE65 in epidermal keratinocytes. Colocalization of plasma retinol-binding protein with RPE65 and affinity binding suggest a direct interaction of RPE65 with plasma retinol-binding protein in cultured human keratinocytes that might be involved in retinoid uptake of keratinocytes.

Numerous reports have indicated that the biological activity of all-trans retinoyl beta-glucuronide (RAG) is similar to that of all-trans-retinoic acid (RA), but without the toxic side effects of RA. In the present series of studies, we report new findings that support the contention that RAG can function as a nontoxic substitute for RA in a variety of clinic settings. One study on the effects of s.c. injected graded doses of RA and RAG (20-480 micromol/kg BW) into pregnant Sprague-Dawley rats showed that any differences between RAG and RA could be observed only at the highest dose levels of 360 and 420 micromol/kg BW, with RAG being much less toxic than RA. Similarly, daily topical application of RAG (0.16-1.6%) and RA (0.1-0.5%) to shaved swine dorsal skin for six mo resulted in redness and scabbing in RA-treated patches, and to a lesser extent in 1.6% RAG-treated, but not in other RAG-treated patches. Histological scores were significantly higher in the dermis and epidermis of RA-treated pigs than in RAG-treated pigs. Studies to document the pharmacokinetics of chronically administered RAG in mice indicated that, unlike RA, sustained blood levels of parent retinoid (RAG) can be achieved during at least 2 mo of daily administration. Another investigation to study the effects of RAG on the development and growth in nude mice of tumors derived from the human neuroblastoma cell line LA-N-5 showed that s.c. injection of RAG (30 micromol/kg BW) reduced tumor formation when the retinoid was first administered 3 d before tumor injection and continued daily for 30 d thereafter. In established tumors, RAG was shown to inhibit progressive tumor growth, the antitumor effects of RAG being comparable with RA. However, with RAG, as opposed to RA, there were no significant adverse physical side effects. Based on the results of these series of studies along with ample published reports over the last 15 y, we conclude that RAG may be a safe and effective alternative to RA and some other retinoids that are presently being utilized in the clinic.

All-trans retinoic acid (tRA, or tretinoin) can be metabolized through stereoisomerization to 13-cis retinoic acid (13-cRA) in vivo. We have developed a simple, sensitive and accurate method for analyzing tRA and 13-cRA in plasma with the addition of N-ethylmaleimide (NEM) and Vitamin C (Vit. C) to prevent the interconversion of cis/trans retinoic acid. All-trans RA, 9-cRA, and 13-cRA were well separated from each other in plasma by using a C18 precolumn and a column with a gradient solvent system of mobile phases A and B at a flow rate of 1.0 ml/min. In addition, thermal stability of tRA and cRA in plasma during the sample preparation under the temperature of 0 and 25 degrees C were studied. Our results showed that (1) the interconversion ratios (%) (cRA/tRA and tRA/cRA) were decreased with the addition of NEM and Vit. C and the minimum concentrations of NEM and Vit. C to inhibit the interconversion were 50 and 150 microM, respectively, (2) higher concentrations of NEM and Vit. C were required to prevent the interconversion at higher temperature, (3) the precision and accuracy of calibration curve with various concentration of tRA (1-1000 ng/ml) and 13-cRA (5-800 ng/ml) in plasma showed good linearity (r(2)=0.9992 and 0.9994), and between-day errors expressed by coefficient of variation (CV, %) for tRA and 13-cRA which were both less than 5.6%, (4) the mean recovery of the analytes were 78-94% for tRA and 80-92% for 13-cRA at concentration range from 1 to 1000 ng/ml and 5 to 800 ng/ml, respectively, and (5) the limit of quantitation of tRA and 13-cRA were 1 and 5 ng/ml, respectively. In addition, the HPLC method had been successfully applied to the tRA pharmacokinetic study in two hepatoma patients after receiving 45 mg/m(2) per day orally. Thus, our results suggest that the HPLC method for analyzing tRA and 13-cRA in plasma with the addition of NEM and Vit. C is a simple, sensitive and accurate method.

Vitamin A is a well-established teratogen in all animal species. A number of case reports also suggest a teratogenic potential of vitamin A in humans. A possible teratogenic risk of dietary liver vitamin A intake, the kinetics of vitamin A and its metabolites in humans after intake of either a vitamin A supplement or a liver meal have been studied. Major differences were described for the kinetics of all-trans-retinoic acid (all-trans-RA), which occurred at much higher concentrations after supplementation than after liver consumption. Therefore, we investigated whether the intestine may be responsible for the differences in vitamin A metabolism after supplementation or liver feeding. We found that cytosolic fractions of porcine enterocytes oxidized retinol to all-trans-RA in vitro with a K(m) of 94-96 micromol/L and a V(max) of 7.9-8.6 pmol/(min x mg protein). In an in vivo approach, the portal vein and the central vein (external jugular vein) of a pig were cannulated. In two subsequent experiments, the pig was given a vitamin A supplement or liver. Plasma samples were taken from portal and central veins. Comparison of retinoid levels in these veins indicated that all-trans-RA was already formed from supplemental vitamin A in the intestine and released into the systemic circulation. Two major metabolic pathways were additionally present in the pig, leading to the formation of glucuronides of all-trans-RA and retinol itself. Our results indicate that intestinal metabolism contributes to the elevated levels of all-trans-RA in the systemic circulation after supplementation with vitamin A, but not after consumption of liver.

All-trans-retinoyl-beta-D-glucuronide (RAG) is an endogenous active metabolite of all-trans-retinoic acid (ATRA). In the present study, the pharmacokinetics of RAG was examined after the administration of a single intravenous does (5, 10, or 15 micromol/kg) and of multiple daily intravenous doses (5 micromol/kg) to rats for 8 days. The plasma concentrations of RAG and ATRA were measured by a reverse-phase HPLC method. A rapid distribution phase of approximately 1 h was observed in all of the rats after single or multiple doses. Thereafter, RAG was eliminated through a first-order process, in accord with a typical two-compartment first order pharmacokinetic profile. After single intravenous doses, the AUC of RAG increased proportionally with the dose and the clearance remained unchanged within the tested doses. There was no statistical significant difference in distribution rate constants from central compartment to peripheral compartment (K(12)) and from peripheral compartment to central compartment (K(21)) between different doses. However, as the dose increased from 5 micromol/kg to 10 micromol/kg, the volume of distribution at the steady state (V(ss)) and the volume of peripheral compartment (V(p)) decreased significantly (p < 0.05) from 1.290 +/- 0.269, 0.928 +/- 0.232. L/kg to 0.961 +/- 0.149, 0.647 +/- 0.107 L/kg, respectively. V(ss) and V(p) at a dose of 15 micromol/kg (0.924 +/- 0.187, 0.698 +/- 0.165 L/kg) were not significantly different from that at 10 micromol/kg. Thus, RAG might saturate the tissue-binding sites at higher doses. ATRA was detected as a metabolite of RAG at low levels (usually < 0.05 microM) only in the first 2 h after intravenous administration. RAG clearly was not extensively hydrolyzed to ATRA in our study. After multiple daily intravenous administration of RAG, the clearance (Cl) and the elimination rate constant (K(10)) remained unchanged (p > 0.05), indicating that long-term daily administration of RAG did not induce its accelerated metabolism. However, K(12), V(p), and V(ss) declined significantly (p < 0.05) from 1.67 +/- 0.54 h(-1), 0.928 +/- 0.232 L/kg, and 1.290 +/- 0.269 L/kg to 0.96 +/- 0.48 h(-1), 0.494 +/- 0.147 L/kg, and 0.818 +/- 0.187 L/kg, respectively. Therefore, long-term daily dosing of RAG seemed to decrease its distribution profile. Although the AUC of RAG did not change significantly after multiple dosing, the AUC of ATRA after RAG dosing significantly declined (p < 0.05) from 0.032 +/- 0.019 microM x h to 0.010 +/- 0.006 microM x h. The decline in the AUC of ATRA might reflect an increase in its uptake by tissue and/or in its metabolism. Because enhanced clearance is not associated with RAG after multiple administrations, RAG could be considered as an alternate to ATRA in appropriate clinical applications., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association)

Although advanced renal-cell carcinoma (RCC) responds poorly to standard therapies, phase I-II trials have shown activity for combinations of interferon-alpha2b (IFN) with a retinoid. Alitretinoin (9-cis RA) is an endogenous retinoid with high binding affinity for both RAR and RXR receptor families. This phase I-II study enrolled 38 patients with RCC in a dose-escalation study of tolerability, pharmacokinetics (PK), and efficacy of twice daily oral 9-cis RA with subcutaneous IFN. In contrast to studies with similar doses of daily 9-cis RA, PK studies found a consistent reduction in 9-cis RA concentrations of about 50% after multiple b.i.d. doses of 30 or 50 mg/m2, independent of cotreatment with IFN. In the phase I portion, toxicities included systemic symptoms typical of IFN and biochemical abnormalities previously associated with retinoids. Two patients experienced dose-limiting toxicity at 50 mg/m2 b.i.d. of 9-cis RA, thus the recommended phase II dose was 30 mg/m2 b.i.d. One of twenty-six evaluable patients achieved a durable objective partial remission, and repeated dosing with this regimen was poorly tolerated. This combination of retinoid and interferon is not recommended for further study in RCC.

all-trans-Retinoic acid (ATRA, vitamin A acid, or tretinoin) is a potent chemotherapeutic agent for the treatment of acute promyelocytic leukemia (APL). Its poor aqueous solubility not only affects its oral absorption but also prevents it from forming an aqueous parenteral formulation. Recently, we developed a water-soluble formulation of ATRA with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD). In present study, this formulation was tested in Sprague-Dawley rats. Kinetic study of ATRA was carried out after oral or intravenous administration. Though there were no statistical differences in any of the estimated pharmacokinetic parameters between ATRA sodium salt and HPbetaCD-based ATRA after intravenous administration, inclusion of ATRA into HPbetaCD was found to greatly improve the oral absorption of ATRA., (Copyright 2000 Wiley-Liss, Inc.)

The intracellular concentration of many steroids and xenobiotics is influenced by the membrane protein P-glycoprotein (Pgp). It has been inferred that the intracellular retention of many drugs that upregulate Pgp or modulate Pgp function might also be affected by Pgp. However, the ability of Pgp to influence the translocation of these drugs needs to be established to understand Pgp's influence upon their pharmacological effect. We utilized two approaches to determine the interaction of several agents with Pgp: (a) an in vitro system, LLC-PK1 cell lines and derivative LLC cell lines stably expressing on the apical membrane either mouse mdr1a or human MDR1 Pgp grown as polarized epithelium in transwell culture to measure translocation of radiolabeled drugs; and (b) an in vivo system, mdr1a nullizygous and wild-type animals, to compare the contribution of Pgp to in vivo distribution of radiolabeled drugs. In combination these complementary approaches identified erythromycin as a drug whose intracellular retention is influenced by Pgp, while the intracellular accumulation and tissue distribution of retinoic acid and benzo(a)pyrene were unaffected by Pgp., (Copyright 1998 Academic Press.)

A novel topical tretinoin gel formulation containing a patented TopiCare Delivery Compound, polyolprepolymer-2 (PP-2), was shown to significantly reduce local irritation relative to a marketed tretinoin gel preparation while maintaining clinical efficacy in the treatment of acne. Several in vitro percutaneous absorption studies were conducted with 0.025% tretinoin as a model compound to determine the possible mechanism of action of PP-2 on drug delivery into and through human cadaver skin. Results of these studies have repeatedly shown that a new topical gel formulation containing PP-2 significantly reduces tretinoin penetration while potentially enhancing epidermal deposition compared with a commercial topical gel preparation at the same tretinoin concentration. These studies further support a mechanism of action whereby PP-2 serves as a retentate for drug delivery by formation of a liquid reservoir of polymer and solubilized drug on the skin surface and in the upper layers of the skin, thereby modifying delivery of tretinoin into and through skin. This reservoir of drug and polymer was established within 15 min after topical application, and tretinoin was shown to be highly associated with PP-2. These in vitro findings provide a model by which a new tretinoin gel formulation containing PP-2 reduces irritation relative to a commercial tretinoin gel while maintaining clinical efficacy in the treatment of acne vulgaris.

A gradient reversed-phase high-performance liquid chromatographic technique is described for the easy separation and quantification of some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and their corresponding 4-oxometabolites, in plasma. The method involved a diethyl ether-ethyl acetate (50:50, v/v) mixture extraction at pH 7 with acitretin and 13-cis-acitretin as internal standards. A Nova-Pak C18 steel cartridge column was used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5% tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min. The gradient composition was (only the percentages of solvent B are mentioned): I, 25% solvent B at the time of injection; II, 12% solvent B at 11 min until min; III, 25% solvent B and maintenance of 25% solvent B for 10 min until a new injection. Total time between injections was 40 min. Detection was by absorbance at 350 nm. The precision calculated for plasma concentrations ranging from 2 to 250 ng/ml was better than 15% and the accuracy was less than 12%. The linearity of the method was in the range of 2 to 400 ng/ml of plasma. The limit of quantification was 2 ng/ml for each of the compounds. The HPLC method was applied to plasma specimens collected from animals receiving single dose administrations of all-trans-retinoic acid, 13-cis-retinoic acid and 9-cis-retinoic acid.

All-trans-retinoic acid (RA) is used successfully in the treatment of acute promyelocytic leukaemia (APL), although unexplained relapses occur in many of the patients. Pharmacokinetic studies may help in understanding the mechanism of resistance to RA and a simple and rapid procedure for its determination in biological samples may be advantageous. A high-performance liquid chromatographic procedure is described, involving one-step extraction of RA from plasma, isocratic elution from a reversed-phase column (LiChrosorb RP-18, 5-microns particle size) and UV detection at 340 nm. The calibration graph is linear over a wide range and the limit of detection is approximately 10 ng/ml, using 0.5 ml of human plasma. The method is selective for RA, accurate and robust and thus suitable for the routine analysis of plasma samples from patients undergoing RA therapy. Analysis of plasma in a patient on RA therapy (45 mg/m2 per day) confirmed that during continuous treatment with RA the drug plasma concentrations are markedly lower at the time of relapse than on the first day of treatment.

It has been shown that all-trans retinoic acid (ATRA) at doses of 45 to 100 mg/m2/d induces complete remission (CR) of acute promyelocytic leukemia (APL) by a differentiation process. To date, ATRA dose-ranging studies have not yet been evaluated. Thus, we initiated in May 1990 a multicenter study with ATRA at a lower dose of 25 mg/m2/d until CR. Thirty patients with APL were treated with ATRA, of whom 12 were previously untreated, 14 were in first relapse, and 4 had failed after conventional first induction chemotherapy. Twenty-four of 30 achieved CR, 3 failed, and 3 died before day 30. Median time to CR was 45 days. Hyperleucocytosis (14 to 43 x 10(9) white blood cells per liter) was observed in 9 patients between days 10 and 23. Clinical complications that may have been related to the retinoic acid syndrome were observed in 8 patients, of whom 3 died. Pharmacokinetics studies were performed in 5 patients. Peak plasma concentrations and mean area under the concentration-time curve were not lower than previous levels obtained under the 45 mg/m2 dose. Overall, our study shows that there is no difference in terms of therapeutic efficacy, triggering of hyperleukocytosis, or retinoic acid syndrome and pharmacokinetic results with ATRA at 25 or 45 mg/m2/d.

A combination of 4-hydroxyanisole (4HA) and all-trans retinoic acid (TRA) was found to synergistically cause moderate to complete depigmentation of Yucatan swine skin. Two hyperpigmentation models were used: Natural dark-skinned swine, a potential model for melasma-like disorders, and ultraviolet light-stimulated hyperpigmentation, a model of solar lentigines. Test materials were applied twice daily, 5 d/week, to dorsal flank skin. Application sites were graded at weekly intervals for skin color using a 0 to 4 grading scale. After 8 weeks of treatment of naturally dark swine skin, a combination of 2% 4HA and 0.01% TRA produced grade 2 hypopigmentation (definite but moderate hypopigmentation). In contrast, 2% 4HA alone or 0.01% TRA alone did not produce significant hypopigmentation. After cessation of treatment, the 4HA/TRA-treated sites reverted to normal color within 7-12 weeks. The 4HA/TRA combination completely reversed the hyperpigmentation induced by ultraviolet light after 8 weeks of treatment. In vitro skin-penetration studies using hairless mouse and human skin show that skin penetration of 4HA was not significantly affected by adding 0.01% TRA. These data suggest that the observed synergy is not due to enhanced bioavailability of 4HA. We have demonstrated that combining low concentrations of 4HA and TRA results in effective skin lightening without causing irreversible depigmentation and with minimal local skin irritation.

Topical bioavailability of drugs incorporated in liposomes is not well known. We compared the skin penetration of tretinoin in liposomes and in a classical alcoholic gel. [3H]Phosphatidylcholine dipalmitoyl (DPPC) and [14C]tretinoin (0.14%) were incorporated in the phospholipidic phase of the liposomes, and [14C]tretinoin was incorporated in a gel for comparison. Skin absorption was studied in vitro with Franz cells. In vivo distribution in cutaneous structures was studied according to Schaefer's method. Liposomes impregnated the stratum corneum, with a partial dissociation between tretinoin and phosphatidyl-choline dipalmitoyl. In dermis, tretinoin diffused alone. Tretinoin release seemed to be controlled, and steady state was reached later with liposomes than with gel. This phenomenon was linked with a significantly reduced absorption (1.60% for liposomes versus 3.09% for the gel) and higher retention in epidermis (mainly stratum corneum) and dermis (41 and 13%, respectively, with liposomal form versus 18 and 8%, respectively, with gel form). This study clearly shows that, compared with the gel, the liposome formulation tends to improve the local effect of tretinoin in the skin and decrease the systemic absorption.

The pharmacokinetics of three 13-cis-retinoic acid formulations were studied after intraperitoneal (ip) administration to rats. Rats were given ip injections of 2.5 mg of 13-cis-retinoic acid per 360 g of body weight; the drug was administered as an alkaline solution, suspended in corn oil, or as a mixture with polysorbate 80. The alkaline solution was also given intravenously (iv) via the tail vein as a control. The mean elimination rate constant, calculated from data from iv administration, was 0.72 +/- 0.088 h-1 (r = 0.988). The peak concentration in plasma and the time to reach this maximum were 14 mg/L and 0.5 h, 22 mg/L and 2 h, and 10 mg/L and 1 h for the drug administered as an alkaline solution, suspended in corn oil, and as a mixture with polysorbate 80, respectively. The areas under the concentration-time curve (concentration in plasma versus time) were 34.9 +/- 8.78 mg.h/L for the iv dose and 34.1 +/- 9.97, 62.4 +/- 32.3, and 25.9 +/- 12.0 mg.h/L for the ip doses of alkaline solution, suspension in oil, and mixture with polysorbate 80, respectively. Because of the rapid increase of concentration in plasma, which is identical to that of the iv profile, and the ease of its handling and preparation, the ip administered alkaline solution is the preferable formulation.

Conjugation reactions, such as the formation of sulfates, glucuronides and amino acid derivatives of biologically active compounds, are classically considered as detoxication processes. In essence, common nonpolar compounds are rendered less active by the formation of water-soluble metabolites, which then can readily be excreted in the urine. In the past decade, however, it has become clear that some natural metabolites become more active rather than less so by the formation of such conjugates. Several compounds in this special group will be considered in this article; namely, glucuronide and hydroxyphenylamide conjugates of retinoic acid, glutathione and cysteine conjugates of halogenated hydrocarbons, glucuronide conjugates of various xenobiotics and polyglutamates of folic acid and its inhibitors. The metabolism, biological activity and toxicity of the parent molecule is profoundly affected in complex ways by such reactions. Thus, the historical concept of conjugation reactions as general detoxication processes is no longer tenable.

A reliable analytical method has been developed for measurement of 13-cis- and all-trans-acitretin (Neotigason) in human plasma by normal-phase high-performance liquid chromatography, with ultraviolet detection. Human plasma was obtained after centrifugation of whole blood samples and deproteinized by ethanolic denaturation. After liquid-liquid extraction with water-n-hexane, and aliquot ws chromatographed on a silica column using isocratic elution with n-hexane-methylsalicylate-acetic acid (200:18:0.6, v/v). The wavelength was set at 360 nm, and for plasma samples a limit of quantification of 3-4 ng/ml was obtained. All manipulations were carried out under dim light conditions to prevent photoisomerization.

The influence of irritant contact dermatitis on percutaneous penetration was investigated for four 14C-labeled compounds with diverse physicochemical properties: hydrocortisone (HC), indomethacin (IM), ibuprofen (IB), and acitretin (AC). Hairless guinea pigs were pretreated in vivo for 24 h with either 0.5% sodium lauryl sulfate (SLS) to induce irritant contact dermatitis or with water (controls). Twenty-four hours after pretreatment animals were sacrificed. Percutaneous penetration was then measured using in vitro diffusion cells and the removed (pretreated) skin. The following parameters were determined: cumulative amount of compound penetrated, steady state flux, lag time, and permeability coefficient, skin concentration per unit area, and the relative amount of drug remaining in the skin (as a percentage of the cumulative amount of compound penetrated through the skin). SLS pretreatment resulted in moderate irritant dermatitis in all animals and increased in vivo transepidermal water loss 4.5 times. Flux was increased in SLS-pretreated skin as compared with controls for all four compounds, with the greatest enhancement for hydrocortisone (HC) (5.9 times), followed by indomethacin (IM) (4.6 times), ibuprofen (IB) (3.9 times), and acitretin (AC) (3.4 times). Skin concentrations increased to a smaller degree from 1.6 times (IB) and 2.6 times (HC) to 3.4 times (IM). However, AC skin concentrations were not different between the two groups. Thus, percutaneous penetration parameters were equivocally influenced by SLS-induced irritation. Increased skin concentrations were paralleled by even higher increases in flux.

A single application of 17 micrograms/kg or 8.7 mg/kg all-trans-[10,11-3H2]-retinoic acid dissolved in acetone to shaved dorsal hamster skin resulted in rapid absorption and dose-dependent rates of elimination. An equation describing a two-compartment open model with a very brief lag time and first-order uptake and elimination was used to describe the central plasma compartment kinetics. Unchanged all-trans-retinoic acid represented less than or equal to 4% of the total circulating radio-activity. Peak circulating concentrations of parent all-trans-retinoic acid were less than those observed after an equivalent oral dose, but prolonged absorption from the skin appears to contribute to high total bioavailability of topical retinoid. Topical administration to intact skin of up to three consecutive doses of 10.5 mg/kg/d all-trans-retinoic acid or a single 5 mg/kg dose of etretinate (Ro 10-9359) during a critical stage of embryogenesis in hamsters caused erythema and/or dose-dependent epidermal hyperplasia at the site of application, but failed to induce a significant teratogenic response. Topical application of 0.01-1.0 mg/kg arotinoid Ro 13-6298 resulted in dose-dependent mucocutaneous toxicity and an increase in the numbers of dead embryos and malformed offspring. The marked skin toxicity and attenuated concentrations in maternal blood, compared to the oral route, limit the amounts of retinoid that can reach the hamster embryo. It is thus more important to compare the retinoid systemic values (absorbed dose) than it is to compare the oral or topical (applied) dose, when interpreting the results of conventional teratogenicity bioassays. The data suggest that in the human it is skin toxicity that limits the amounts of retinoid that can be applied and subsequently reach the embryo. In the rodent, overt skin toxicity under continued dosing could increase the amounts of retinoid penetrating the skin and reaching the embryo.

Normal subjects received fenretinide (HPR), 200 mg/d, on three schedules. Schedule 1 was treatment for 28 d. Schedule 2 consisted of 14 d of treatment, 3 d hiatus, and a second drug course of 14 d, 10,000 IU vitamin A was administered during the 3-d hiatus. Schedule 3 was 14 d of treatment followed by a rest period of 7 d and then 14 d of treatment. Increase in plasma HPR was accompanied by an even higher increase in the metabolite N-(4-methoxyphenyl)-all-trans-retinamide (MPR). The administration of HPR was associated with a significant reduction in retinol-binding protein (RBP), which returned to pretreatment values after the drug treatment was discontinued. Reduction of plasma retinol was also observed. Use of interrupted schedules with resting periods of 3 and 7 d changed HPR, MPR, and RBP concentrations in plasma. Addition of vitamin A did not affect the pattern of the measured variables in the plasma.

Etretinate accumulates in adipose tissue; this appears to account for its long terminal elimination phase in psoriatic patients. The purpose of the present study was to investigate the pharmacokinetic profile of etretinate and acitretin in a genetically obese rodent model, the Zucker rat. Pairs of obese and lean Zucker rats were dosed intravenously (0.5 mg/kg) and blood samples were collected. Plasma concentrations of etretinate and its major metabolites, acitretin and the cis isomer of acitretin (isoacitretin), were assayed by HPLC. The systemic clearance (CLs) of etretinate and the formation clearance (CLf) of the metabolite (acitretin) were lower in the obese rats (132 and 62.4 mL/min, respectively) compared with their lean littermates (197 and 126 mL/min, respectively). The remaining metabolic clearance (CLd) was identical for the lean and obese animals (70.9 and 69.9 mL/min, respectively). The ratio of metabolite-to-parent drug area under the plasma concentration-time curve (i.e., acitretin:etretinate) in the obese animal was less than that value in the lean animals (0.348 versus 0.811, respectively) following the administration of etretinate. Despite a doubling in the mean value (204 versus 87.9 mL), no statistically significant differences in the volume of distribution term for etretinate (Vdss) was observed in the obese animals, due to the large interanimal variability. The terminal phase half-life (t1/2) was significantly longer in the obese rats (3.52 versus 1.25 h). Following acitretin administration, no statistically significant differences were observed between the obese and lean animals for any of the parameters (CLs, Vdss, MRT, t1/2) of acitretin.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic acid, an endogenous metabolite of vitamin A (retinol), possesses striking biological activity akin to a morphogen in developing and regenerating vertebrate limbs. Systemic administration of retinoic acid (RA) to pregnant mammals during the period of limb organogenesis invariably results in dose-dependent dysmorphogenesis. In an attempt to uncover the mode of action of RA in the developing limb bud we analyzed, by HPLC methods, the levels of RA and its metabolic precursor, retinol, in embryonic mouse tissues prior to and following maternal exposure to a teratogenic dose of RA. Detectable levels of both RA and its isomer 13-cis-retinoic acid were found in the limb buds of Day 11 mouse embryos (40 +/- 2 somites). Although retinol was the major retinoid found in ethanolic extracts of either whole embryo or the limb buds, the latter is enriched in RA compared to the whole embryo. This indicated either a higher degree of retinol metabolism or a sequestration of RA in the limb bud compared to the rest of the embryo at this stage of development. A study of the time course of retinoid levels in treated embryos showed that changes occur rapidly, are stable for several hours, and then begin to return to pretreatment levels. After a maternal dose of 10 mg/kg RA, which resulted in a mild degree of limb anomalies, peak RA levels in the limb bud increased 50-fold over the endogenous level; a full 300-fold increase was found after a 100 mg/kg dose which results in 100% incidence of phocomelia. Interestingly, a dose-dependent depression in retinol levels was observed after RA treatment both in maternal plasma as well as the embryo. Studies are in progress to trace the intracellular disposition of both retinol and RA as well as any further active metabolite of RA in the limb buds and other embryonic tissues.