Arotinoid Acid Research Articles

Synthesis of arotinoid acid and temarotene using mixed (Z)-1,2-bis(organylchalcogene)-1-alkene as precursor

An efficient and novel total synthesis of the two bioactive retinoids temarotene and arotinoid acid (TTNPB) is described. The key steps in this process include the regio and stereoselective hydrotelluration of thioacetylene 9 and Te/Li transmetalation of mixed (Z)-1,2-bis(organylchalcogene)-1-alkene (Z)-3. The subsequent reaction involving the β-phenylthio vinyl lithiated intermediate 10 with dimethyl sulfate gave the (E)-vinyl sulfide 11. The Ni+2 cross-coupling of 11 with the corresponding phenylzinc bromide and p-oxazoline phenylzinc bromide 12 afforded the respective temarotene 2 and retinoid-oxazoline substituted 13. Finally, compound 13 was deprotected with HCl to furnish arotinoid acid (TTNPB) 1.

Determination of arotinoid acid in human plasma by liquid chromatography–tandem mass spectrometry

Arotinoid acid (Ro 13-7410) is the third generation of synthetic retinoid, which was developed for the treatment of psoriasis and other hyperkeratotic skin disorders. The therapeutically active dose is less than 0.5 μg/kg body weight/day. To investigate the pharmacokinetics of arotinoid acid, a sensitive and selective liquid chromatographic–tandem mass spectrometric method (LC–MS/MS) for the determination of arotinoid acid in human plasma was developed and validated. The sample processing was carried out in the dark to minimize photodegradation of the analytes. Arotinoid acid and the internal standard (IS), acitretin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was achieved on a Zorbax Extend C 18 column (150 mm × 4.6 mm, i.d., 5 μm) using methanol:acetonitrile:5 mM ammonium acetate (48:32:20, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. The detection was carried out in multiple reaction monitoring (MRM) mode via negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 37.1 pg/mL with intra-day and inter-day precision (RSD) of 8.7% and 8.5%, and accuracy (RE) of 2.0%. Inter-day and intra-day RSD for three quality control levels (QCs) across validation runs were less than 11.0% and the accuracy ranged from 1.9% to 3.3%. The validated LC–MS/MS method was applied to a phase I clinical pharmacokinetic study after a single oral administration of 40 μg arotinoid trometamol to healthy subjects.

Rétinoïdes par voie générale (acitrétine-isotrétinoïne)

Arotinoid acid (Ro 13-7410) is the third generation of synthetic retinoid, which was developed for the treatment of psoriasis and other hyperkeratotic skin disorders. The therapeutically active dose is less than 0.5 μg/kg body weight/day. To investigate the pharmacokinetics of arotinoid acid, a sensitive and selective liquid chromatographic–tandem mass spectrometric method (LC–MS/MS) for the determination of arotinoid acid in human plasma was developed and validated. The sample processing was carried out in the dark to minimize photodegradation of the analytes. Arotinoid acid and the internal standard (IS), acitretin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was achieved on a Zorbax Extend C18 column (150 mm × 4.6 mm, i.d., 5 μm) using methanol:acetonitrile:5 mM ammonium acetate (48:32:20, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. The detection was carried out in multiple reaction monitoring (MRM) mode via negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 37.1 pg/mL with intra-day and inter-day precision (RSD) of 8.7% and 8.5%, and accuracy (RE) of 2.0%. Inter-day and intra-day RSD for three quality control levels (QCs) across validation runs were less than 11.0% and the accuracy ranged from 1.9% to 3.3%. The validated LC–MS/MS method was applied to a phase I clinical pharmacokinetic study after a single oral administration of 40 μg arotinoid trometamol to healthy subjects.

Prurigo: diagnosis and management.

Prurigo is a condition of nodular cutaneous lesions that itch (pruire) intensely. Although the acute form can be caused by insect stings, most of the subacute and chronic forms appear to be idiopathic. Toxic agents deposited in the skin by exogenous factors such as parasites, bacteria, or topically or orally administered drugs can induce itch. In susceptible individuals, physical mechanisms such as UV light can induce changes in epidermal innervation that result both in itch generally and in prurigo lesions. Prurigo is sometimes associated with atopy, pregnancy, internal diseases, malabsorption, or malignancy. Some forms of prurigo may be secondary to scratching. Emotional factors can also influence the perception of itch and induce prurigo by provoking scratching. These are the various specialized forms of prurigo, and there are certain others, such as prurigo pigmentosa, that have some ethnic preference. Topical treatments by corticosteroids, coal tar, bath photochemotherapy, UVB, cryotherapy, or capsaicin, as well as systemic regimens involving use of psoralen + UVA (PUVA), erythromycin, arotinoid acid, cyclosporine, chloroquine, dapsone, minocycline, naltrexone, azathioprine or thalidomide are used for the treatment of this condition. Psychotherapeutic agents to treat problems of mood that deteriorate prurigo are also prescribed. Combined sequential treatments for generalized, therapy-resistant cases need to be tailored to the exacerbations that occur and to provide maintenance treatment in order to enable the patient to withstand the intolerable itch.

RETINOIC ACID RECEPTOR-GAMMA-SELECTIVE RETINOIDS EXERT ANTIPROLIFERATIVE EFFECTS ON HUMAN-MELANOMA CELL-GROWTH IN-VITRO

Malignant melanoma is a cancer with dramatically increasing incidence and mortality. Alternative approaches for therapy of disseminated malignant melanoma are urgently needed. In order to develop new alternatives for therapy of metastatic melanoma, we tested the biological activity of eight synthetic retinoids with high affinity and/or selectivity for the retinoic acid receptors (RAR) alpha, beta and gamma in comparison to all-trans retinoic acid, arotinoid acid and CD 367 in four human melanoma cell lines. The melanoma cell growth was not affected by most retinoids tested, however, two of the synthetic substances with high RAR-gamma selectivity (CD 437 and CD 2325) showed a dose-dependent antiproliferative effect on all melanoma cell lines tested with IC50 values between 10(-6) and 10(-7) M. A structurally closely related compound, CD 2398, lacking the carboxyl-terminal group and therefore exhibited no RAR-binding did not change melanoma cell growth behaviour. We demonstrate the expression of RAR-alpha, RAR-beta and RAR-gamma, in four human melanoma cell lines by polymerase chain reaction. Taken together, these results suggest that binding and stimulation of the RAR-gamma receptor might be a major factor in growth inhibition of human melanoma cell by retinoids in vitro. Although several of the compounds altered the cell surface expression of defined melanocytic differentiation markers, the correlation to changes in melanin content was poor. Furthermore, five out of eight retinoids modulated HLA-DR expression on human melanoma cells. The potent growth inhibitory effect of the RAR-gamma selective retinoids as well as their immunomodulating capacities opens an interesting alternative for new antiproliferative and immunomodulatory strategies in the therapy of metastatic melanoma.

Simple Assays of Retinoid Activity as Potential Screens for Compounds That May Be Useful in Treatment of Psoriasis

Human epidermal cell cultures were used to study the effects of retinoids on keratinocyte differentiation. Keratin profiles were studied by quantitative gel electrophoresis of culture extracts, whereas the extent of envelope formation was assessed in an enzyme-linked immunosorbent assay (ELISA) using an antibody that specifically recognizes keratinocyte envelopes. Exposure of cultures to a variety of different retinoids produced both dose-dependent decreases in keratin 16 with consequent increases in the keratin 14: keratin 16 ratio, and a decrease in envelope formation. The order of activity in both assays was similar: arotinoid ethyl ester (Ro 13-6298) greater than or equal to arotinoid acid (Ro 13-7410) much greater than all trans retinoic acid (Ro 1-5488) greater than acitretin (Ro 10-1670) greater than or equal to etretinate (Ro 10-9359), the only difference being that acitretin was slightly more active than etretinate in the keratin assay whereas these retinoids were equi-active in the envelope assay. Analysis of the lesional keratins of psoriasis patients showed that etretinate caused a reduction in keratin 16 and an increase in the keratin 14:keratin 16 ratio, although the magnitude of these changes and their correlation with clinical improvement was variable. As the in vitro assays reported here are simple and quick, they allow rapid screening of compounds for retinoid-like activity.

Stimulation by Retinoids of Tissue-Type Plasminogen Activator Secretion in Cultured Human Endothelial Cells

Retinoic acid induces tissue-type plasminogen activator (t-PA) but not plasminogen activator inhibitor-1 (PAI-1) expression in cultured human umbilical vein endothelial cells (HUVEC). To further investigate the relation between the structure of the retinoids and their ability to induce t-PA synthesis in vitro, 11 analogues were studied in HUVEC culture. The retinoid analogues were classified into one of three groups according to their t-PA-inducing potential. Group 1 showed little induction (0.9- to 1.9-fold after 48 h) at concentrations between 10(-8) and 10(-6) M. Group 2, which includes all-trans-retinoic acid, induced t-PA threefold to fivefold at 10(-6) M but had little effect at 10(-8) M (less than threefold). Group 3, which comprises arotinoid acid (RO-13-7410) and RO-13-6307, induced t-PA antigen secretion fivefold at 10(-8) M. The retinoids of groups 2 and 3 had a terminal carboxyl group and alkyl substitution of the lipophylic head of the retinoid skeleton. The group 3 retinoids also contained an aromatic ring. The t-PA-inducing activity of these third-generation retinoids correlates to some extent with other activities, including regression of papilloma, keratinization in vivo, and clonal inhibition of tumor cell lines in vitro. Some of the retinoids caused a small but significant (up to 1.5-fold at 24 h) increase in PAI-1 antigen secretion. The group 3 retinoids appear to be sufficiently potent inducers of t-PA secretion to warrant further investigation in in vivo animal models.

Anetodermic prurigo nodularis (with Pautrier's neuroma) responsive to arotinoid acid

A woman had symmetrically distributed pruritic nodules for more than 40 years. Microscopically they were characterized by proliferation of nonmyelinated nerve fibers (Pautrier's neuroma) and a total absence of epidermal involvement. Anetoderma developed at the site of former nodules. Her condition responded favorably to repeated retinoid therapy with regression of nodules in those sites in which retinoid dermatitis had developed.

Retinoic acid and synthetic analogs differentially activate retinoic acid receptor dependent transcription

We have developed an assay where the potency of retinoids in retinoic acid receptor (RAR) mediated transcriptional activation can be rapidly evaluated. In this assay hRAR-alpha, hRAR-beta and hRAR-gamma were expressed in CV-1 cells together with a reporter gene containing a retinoic acid responsive element (TRE3-tk-CAT). Concentrations required to obtain half-maximum induction (ED50) of CAT-activity were determined for several retinoids, e.g., all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), arotinoid acid (TTNPB) and m-carboxy-arotinoid acid (m-carboxy-TTNPB, an inactive arotinoid analog). The ED50 values for RA decreased in the order of RAR-alpha (24 nM) greater than RAR-beta (4.0 nM) greater than RAR-gamma (1.3 nM), while the ED50 values for TTNPB and 13-cis-RA decreased in the order of RAR-alpha (6.5 nM, 190 nM) greater than RAR-gamma (2.3 nM, 140 nM) greater than RAR-beta (0.6 nM, 43 nM), respectively. No significant inductions were obtained when cells were treated with m-carboxy-TTNPB, even at 10 microM concentrations. The fold induction of CAT-activity for all compounds tested decreased in the order of RAR-alpha greater than RAR-beta greater than RAR-gamma.

Effect of Retinoids on Hyperproliferation-Associated Keratins K6 and K16 in Cultured Human Keratinocytes: A Quantitative Analysis

The keratin patterns of human epidermal keratinocytes cultured on a 3T3-feeder layer in the presence of 10(-8) M non-aromatic (all-trans retinoic acid and 13-cis retinoic acid) and polyaromatic (arotinoid, arotinoid-sulfone, and free arotinoic-acid) retinoids were analyzed by high resolution one- and two-dimensional gel electrophoresis and immunoblotting. Laser densitometric evaluation of one-dimensional gels allowed to quantitate the changes within the keratin patterns and revealed an increase in the expression of keratins K13, K15, and K19 as induced by both non-aromatic and polyaromatic retinoids, except for the parent compound arotinoid. This would then indicate that such keratinocytes are pursuing a more embryonic type of differentiation. In evaluating the data for the hyperproliferation-associated keratins K6 and K16 we noticed an unexpected result: except for all-trans retinoic acid, these two keratins showed opposite responses. As compared to control cultures, the amount of K6 did generally increase, while K16 was reduced, with arotinoid acid being the most effective retinoid. The apparently uncoupled expression of K6 and K16 appeared also to be concentration dependent when 13-cis retinoic acid at concentrations of 10(-9), 10(-8), and 10(-7) M was analyzed. Considering the overall antiproliferative potency of retinoids, we therefore conclude that K16 alone, rather than the pair K6/K16, should be regarded as a proliferation-related keratin and as such may be used as a sensitive marker to evaluate keratinocyte proliferation.

Retinoids and Keratinocyte Differentiation in vitro

Standard retinoids (e.g. etretinate, isotretinoin) are successful in the treatment of a variety of dermatological disorders. However, they are handicapped by an unfavorable ratio between activity and toxicity. In order to find more successful substances new promising compounds were recently synthesized. The aim of this study was to screen new monoaromates (etretin (Ro 10-1670) ), demethyletretin (Ro 12-7310), 13-cis-etretin (Ro 13-7652) and the polyaromates arotinoid (Ro 15-0778), arotinoid acid (Ro 13-7410) and arotinoid ethyl sulfone (Ro 15-1570) for their effect on keratinocyte differentiation in vitro and to compare these results with differentiation data of retinoids currently used clinically. It was found that the second generation of retinoids had a lower antikeratinizing potential than the third generation of retinoids. They markedly inhibited crosslinked keratins and envelope proteins. Arotinoid acid was found to be the most potent derivative. In contrast to this, differences with regard to the synthesis of keratohyalingranule macroaggregates were detected between the retinoid derivatives. Arotinoid acid and arotinoid sulfone stimulated this protein fraction, whereas arotinoid left this fraction uninfluenced. If the data obtained were considered the selection criterion for the future therapeutic application of the new arotinoids in psoriasis therapy, it should be more useful to employ arotinoid acid or arotinoid sulfone rather than arotinoid in clinical psoriasis studies.