Inhibitory effect of homochlorcyclizine on melanogenesis in α-melanocyte stimulating hormone-stimulated mouse B16 melanoma cells.

The histamine receptor H1 antagonist homochlorcyclizine (HC) has been widely used as an antihistamine agent for the treatment of allergies. However, the effect of HC on skin pigmentation is not known. In the present study, we investigated the inhibitory effect of HC on melanogenesis in mouse B16 melanoma cells. Our results showed that HC inhibited melanogenesis in either α-melanocyte stimulating hormone (α-MSH)- or 3-isobutyl-1-methylxanthin (IBMX)-stimulated B16 cells in a dose-dependent manner. Despite the strong inhibition of melanogenesis by HC, it was surprisingly found that HC did not reduce either cellular or melanosomal tyrosinase activity in α-MSH-stimulated B16 cells. In addition, HC also did not directly inhibit either murine or mushroom tyrosinase activity in the cell-free system. Moreover, western blotting and reverse-transcription polymerase chain reaction (RT-PCR) analyses respectively confirmed that HC did not downregulate levels of tyrosinase protein and its mRNA in α-MSH-stimulated B16 cells. These results clearly demonstrated that HC inhibits melanogenesis of B16 cells by a mechanism other than reduction of the cellular tyrosinase activity. From the present study, HC was proven to be a good candidate as a skin-whitening agent for treatment of skin hyperpigmentation, and this generic drug might be suitable for use in combination with other depigmenting agents due to its unique inhibition mechanism.

The pharmacokinetics and pharmacogenetics of the antiemetic cyclizine in palliative care patients.

CONTEXT: Cyclizine, an antihistaminic antiemetic, is commonly used in palliative care. Its pharmacokinetics have been poorly studied, and its metabolic pathway is unknown but may involve the genetically controlled cytochrome P450 2D6 (CYP2D6). If this is the case, the metabolic ratio of cyclizine to norcyclizine and efficacy/adverse effects may vary between patients according to their CYP2D6 genotype. OBJECTIVES: To deduce the pharmacokinetics and antiemetic/sedative effects of cyclizine and relate these and its metabolic ratio to the CYP2D6 genotype in palliative care patients. METHODS: Palliative care patients initiated on continuous cyclizine subcutaneous (SC) infusions had blood samples taken and efficacy/toxicity scores measured during the approach to steady state. Another group of patients at steady state receiving oral cyclizine had a single blood sample taken. Samples were analyzed to elucidate pharmacokinetic parameters and CYP2D6 genetics. RESULTS: SC dosing group: The median (interquartile range) cyclizine half-life, volume of distribution, and clearance were 13 (7-48) hours, 23 (12-30)L/kg, and 15 (11-26)mL/min/kg, respectively. Nausea and sedation scores were 3.0 (1.2-5.7) and 5.0 (2.6-8.1), respectively, overall and did not vary with genotype (P=0.76 and 0.11, respectively). The median overall metabolic ratio at steady state was 4.9 (3.8-9.2) and did vary with CYP2D6 genotype (P=0.02). Oral dosing group: The median metabolic ratio was 2.1 (1.5-2.9) and did not vary with CYP2D6 genotype (P=0.37). CONCLUSION: Palliative care patients have similar cyclizine pharmacokinetics to those reported in other patient groups. Cyclizine metabolism to norcyclizine may include CYP2D6 as the metabolic ratio varied with CYP2D6 genotype in the SC group.

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

A rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for quantification of cyclizine and its main metabolite norcyclizine in human plasma. Samples were prepared by protein precipitation with acetonitrile and cinnarizine was used as internal standard (recovery >87%). The analytes were eluted from a C8 50 mm×2.0 mm analytical column using a linear gradient of methanol and 0.05% formic acid with a total analysis time of 4 min. Analytes were detected by MS/MS using electrospray ionisation in the positive mode with multiple reactions monitoring (MRM) of the precursor ion/product ion transitions 267.2/167.2 for cyclizine and 253.2/167.2 for norcyclizine. Matrix effects were negligible. Standard curves for cyclizine and norcyclizine were linear (r(2)≥0.996) over the range 2-200 ng/mL, with 2 ng/mL representing the lower limit of quantification. Relative standard deviations were <14% for intra- and inter-day precision and the accuracy was within ±8%. The assay was successfully applied to a clinical study.

Synthesis and in vitro antiproliferative activity of novel 1-benzhydrylpiperazine derivatives against human cancer cell lines.

In order to explore the antiproliferative effect associated with the piperazine framework, several 1-benzhydrylpiperazine derivatives 8(a-d), 9(a-d) and 10(a-h) were synthesized. Variation in the functional group at N-terminal of the piperazine led to three sets of compounds, bearing the sulfonyl, amide and thiourea, respectively. Their chemical structures were confirmed by (1)H NMR, LCMS, IR and elemental analysis. The antiproliferative effect of the compounds were evaluated in vitro using the MTT colorimetric method against one normal cell line (NF-103 skin fibroblast cells) and four human cancer cell lines (MCF-7 breast carcinoma cell line, HepG-2 hepatocellular carcinoma cell line, HeLa cervix carcinoma cell line and HT-29 colon carcinoma cell line) for the time period of 24 h. Among the series, four compounds exhibited interesting growth inhibitory effects against all four cell lines.

Acute eosinophilic pneumonia caused by calcium stearate, an additive agent for an oral antihistaminic medication.

A 70-year-old man was admitted to our hospital because of dyspnea after taking an antihistaminic agent (homochlorcyclizine hydrochloride) for itching. Chest roentgenogram showed infiltration in the left lung field, and laboratory data revealed eosinophilia. Examination of the bronchoalveolar lavage fluid revealed an increased eosinophil count. A drug lymphocyte stimulation test was positive only for calcium stearate, an additive contained in the homochlorcyclizine hydrochloride tablet. The pulmonary infiltration and clinical symptoms subsided after withdrawal of all drugs and initiation of glucocorticoid therapy. Therefore, we concluded that this patient's pulmonary disease was caused by calcium stearate, an additive for an antihistaminic drug. An allergic reaction to a drug's additive material should be considered as a rare cause of drug-induced acute eosinophilic pneumonia.

Effect of (+) or (-) camphorsulfonic acid additives to the mobile phase on enantioseparations of some basic drugs on a Chiralcel OD column.

This paper describes the modification of Chiralcel OD column properties by adsorption of (+) or (-) camphorsulfonic acids (CSAs) used as additives to the mobile phase. The effects on retention, selectivity and efficiency, of adsorption of (+) and (-) CSAs on a Chiralcel OD column were examined. Racemic anti-histamines, anti-malarial and anti-fungal drugs, namely doxylamine, miconazole, sulconazole, hydroxyzine, homochlorcyclizine, methoxypheniramine, cyclopentolate and ephedrine were investigated as chiral tested compounds. All the studied drugs have an amino nitrogen atom in their structure. Only the enantioseparation of ephedrine enantiomers with CSAs alone was studied on the Nucleosil stationary phase, and these results were compared with the results obtained on the Chiralcel OD phase. A new dynamically generated stationary phase, with very good enantioseparation ability towards the studied compounds, was obtained by the adsorption of (-) CSA on the Chiralcel OD column.

Percutaneous absorption of cyclizine and its alkyl analogues.

Cyclizine (I) alkyl analogues (II-IV) were synthesized and their skin permeation parameters evaluated in vitro. It was hoped that these compounds would possess physicochemical properties more favourable for percutaneous delivery than (I). The identification and levels of purity for the compounds were confirmed by mass spectrometry (MS), nuclear magnetic resonance (NMR) spectrometry, and infrared spectrometry (IR) while melting points were determined by an electrothermal digital Bupsilonchi melting point apparatus. Aqueous solubilities (25 degrees C) and partition coefficients were determined and in vitro permeation studies were performed in buffer (37 degrees C) at pH 7.4 over a period of 24 h, using Franz diffusion cells fitted with human epidermal membranes. Generally, the analogues were more lipophilic, but nevertheless possessed higher aqueous solubilities as compared to (I). (II) and (IV) exhibited two- to three-fold increase in aqueous solubility and their melting temperatures dropped by more than 55 degrees C. Compound (III) had similar aqueous solubility to (I), but its melting point dropped by about 35 degrees C. Measured steady-state fluxes indicated that (II) is a far better penetrant (J=6.95 microg/cm(2)/h) of human epidermis than (I). Although fluxes of (III) and (IV) drop off markedly from that of (II), they remained above the flux of (I), which is (0.132 microg/cm(2)/h). In conclusion, (II) was the best skin permeant and also exhibited the highest aqueous solubility and lowest level of crystallinity as compared to (I) and other analogues. (III) and (IV) were more lipophilic. The overall permeation data of this series indicated that the more water-soluble and the lowest melting point compound was the best skin permeant.

Histamine H1-receptor antagonists, promethazine and homochlorcyclizine, increase the steady-state plasma concentrations of haloperidol and reduced haloperidol.

The effects of histamine H1-receptor antagonists, promethazine and homochlorcyclizine, both of which are inhibitors of CYP2D6, on the steady-state plasma concentrations (Css) of haloperidol and reduced haloperidol were studied in 23 schizophrenic inpatients receiving haloperidol, 12 to 36 mg/d, for 2 to 29 weeks. Promethazine, 150 mg/d, in 11 patients and homochlorcyclizine, 60 mg/d, in the others were coadministered for at least 1 week. Blood sampling was performed before and during coadministration of promethazine or homochlorcyclizine and 1 week after the discontinuation, together with clinical assessments by Brief Psychiatric Rating Scale (BPRS) and Udvalg for kliniske undersogelser (UKU) side effect rating scale. The Css (mean +/- SD) of haloperidol and reduced haloperidol during promethazine coadministration (27.6 +/- 24.9 and 8.6 +/- 13.2 ng/mL) were significantly higher than those before the coadministration (12.7 +/- 10.8 and 5.0 +/- 6.0 ng/mL; P < 0.01) or 1 week after the discontinuation (15.6 +/- 14.8 and 5.8 +/- 7.9 ng/mL; P < 0.05). The Css of haloperidol and reduced haloperidol during homochlorcyclizine coadministration (14.9 +/- 8.1 and 6.4 +/- 5.4 ng/mL) were also significantly higher than those before the coadministration (10.9 +/- 7.2 and 3.8 +/- 3.6 ng/mL; P < 0.01) or 1 week after the discontinuation (12.9 +/- 7.4 and 4.8 +/- 4.1 ng/mL; P < 0.05). No change in BPRS or UKU score was found throughout the study. Thus, the current study suggests that coadministration of clinical doses of promethazine and homochlorcyclizine increases the Css of haloperidol and reduced haloperidol via the inhibitory effects on the CYP2D6-catalyzed metabolism of haloperidol and reduced haloperidol.

Biotransformation of cyclizine in greyhounds. 1: Identification and analysis of cyclizine and some basic metabolites in canine urine by gas chromatography-mass spectrometry.

1. The partial in vivo biotransformation of Marezine [(cyclizine.HCl); 1-diphenylmethyl-4-methylpiperazine hydrochloride] in the racing greyhound and the excretion of the unconjugated and conjugated (Phase II) basic metabolites of cyclizine in canine urine are reported. 2. Using copolymeric bonded mixed-mode solid-phase extraction cartridges, the basic isolates from both unhydrolysed and enzyme hydrolysed urine samples were isolated, derivatized as trimethylsilyl ethers and analysed by positive-ion electron ionization gas chromatography-mass spectrometry (EI(+)-GC-MS). Selected samples were analysed by positive-ion methane chemical ionization (CI(+))-GC-MS to aid structure elucidation of the putative metabolites. 3. Cyclizine was the major component excreted in post-administration urine. Five substrate-related basic compounds (M1--> M5) were tentatively identified by EI(+)- and CI(+)-GC-MS. The major Phase I metabolite was identified as norcyclizine [1-diphenylmethylpiperazine] (M1), the other metabolites (M2 --> M5) were tentatively identified as monohydroxylated products based on MS data. 4. Cyclizine and the N(4)-desmethyl metabolite (M1) are excreted unconjugated; the other four hydroxylated metabolites are excreted as Phase II conjugates (glucuronides and/or sulphates). Structures of the putative basic metabolites are presented. At least four other basic metabolites were also detected in post-administration urine, but could not be characterized from GC-MS data. 5. All unhydrolysed post-administration urine samples were analysed by selected ion monitoring EI(+)-GC-MS to quantify cyclizine and norcyclizine (M1) using authentic cyclizine as the analyte and chlorcyclizine as the internal standard. The level of M1 is expressed as 'cyclizine equivalents'. The duration of urinary elimination of cyclizine and M1 was obtained from their excretion profiles. 6. From these studies, cyclizine and norcyclizine (M1) would be the target compounds of choice in the development of screening and confirmatory methods for the detection of cyclizine administration to racing greyhounds. Information on any of the other metabolites may also be of some value for confirmatory analysis.

Biotransformation of cyclizine in greyhounds. 2: N(1)-dealkylation and identification of some neutral and phenolic metabolites in canine urine by gas chromatography-mass spectrometry.

1. The in vivo enzymatic Phase I biotransformation of cyclizine (Marezine in the racing greyhound has been shown to proceed via several different pathways. Aromatic and heterocyclic oxidation and the N(4)-demethylation of cyclizine lead to the formation of unconjugated and conjugated (Phase II) basic metabolites excreted in canine urine. 2. Enzymatic N(1)-dealkylation of cyclizine and its basic metabolites leads to the formation of the neutral and phenolic Phase I metabolites containing the diphenylmethane/methylene substructures. Further, Phase I metabolism of the neutral metabolites could also lead to the formation of several secondary phenolic products. These neutral and phenolic compounds are then excreted as unconjugated and Phase II conjugates in greyhound urine. 3. Following enzymatic deconjugation of selected post-Marezine administration urine samples from two greyhounds, the total aglycones were extracted and separated into neutral/acidic and basic fractions using copolymeric mixed-mode solid-phase extraction cartridges. 4. The neutral/acid isolates were further separated into neutral and phenolic fractions by column chromatography on a lipophilic strong anion-exchanger gel, triethylaminohydroxypropyl Sephadex LH-20 in OH(-) form. 5. The individual neutral and phenolic fractions obtained from the acid/neutral isolate were derivatized as trimethylsilyl ethers and analysed by positive-ion electron ionization gas chromatography-mass spectrometry (EI(+)-GC-MS). 6. Three compounds, diphenylmethane (M1), benzophenone (or diphenyl ketone, M2) and benzhydrol (M3), were identified in the neutral isolates by comparison of their EI(+) mass spectra with authentic standards. At least seven secondary compounds containing the functionalized diphenylmethylene substructure were detected in the phenolic isolates. As no authentic compounds are available, the structures of these putative metabolites (M4--> M10) were elucidated from an interpretation of the EI(+)-GC-mass spectra of their TMS derivatives.

Sensitive high-performance liquid chromatographic determination of cyclizine and its demethylated metabolite, norcyclizine, in biological fluids using coulometric detection.

An accurate, sensitive, selective and reproducible high-performance liquid chromatographic method with coulometric detection for the determination of cyclizine and its inactive demethylated metabolite, norcyclizine, in biological fluids has been developed. The drugs were separated using a custom packed reversed-phase C18 analytical column and phosphate buffer (0.05 M, pH 3)-acetonitrile (7:3) as mobile phase. The dual electrode coulometric detector was operated in the "oxidative-screen" mode with the upstream electrode (detector 1) set at 0.55 V and the downstream electrode (detector 2) set at 0.90 V. Serum and urine samples were prepared for analysis by solid-phase extraction, followed by a simple phase-separation step. The limit of quantitation was 1 ng/ml for both cyclizine and norcyclizine in serum and urine.

Detection of action, inhibition and augmentation spectra in solar urticaria.

BACKGROUND: The action spectrum of solar urticaria varies among cases. In addition, light spectra outside the activating wavelengths can influence the wheal formation in selected patients. OBJECTIVE: To know the mechanisms of light energy, we examined the effect of wavelengths on the skin and the serum factor. METHODS: The patient's skin and serum were exposed to artificial light sources, in vivo and in vitro, respectively. RESULTS: The action spectrum ranged from UVA to visible light (approximately 480 nm). The exposure to longer wavelengths immediately after the exposure inhibited the development of urticaria. Conversely, the irradiation of longer wavelengths before exposure increased the wheal formation. Furthermore, UVB irradiation prior to the exposure of urticaria-eliciting light also increased the urticarial reaction, while postirradiation of UVB had no effect. The patient developed an urticarial wheal at the site of injection of her own serum, which had been previously exposed to the action spectrum in vitro. Preirradiation increased the production of the photosensitizer, while postirradiation revealed no effect. Ultrafiltration techniques showed that the molecular weight of the photosensitizer is more than 300 kD. CONCLUSION: We detected action, inhibition and augmentation spectra in a patient with a severe solar urticaria. Various wavelengths influence the wheal-forming factor in complex interactions.

Effect of first-pass metabolism on enantioselective pharmacokinetics after oral administration of (+)-, (-)- and racemic homochlorcyclizine to rats.

The enantioselective relationship between the pharmacokinetics and hepatic metabolism of homochlorcyclizine hydrochloride (HCZ) was investigated using rats. There were no significant differences in blood concentrations between the three forms after intravenous administration (5 mg/kg) of (+)-, (-)- and racemic HCZ. On the other hand, there were significant differences in the pharmacokinetics between (-)- and (+)-HCZ and between (-)- and racemic HCZ after oral administration (50 mg/kg) of these three forms. The Cmax and AUC0-infinity of (-)-HCZ were lower than those of (+)-isomer and racemate, and its CLo was clearly higher than the others. The (+)-isomer and racemate showed no significant differences in their pharmacokinetic parameters. At a lower dose (10 mg/kg), however, no enantiomeric differences were found in the pharmacokinetic parameters of (+)- and (-)-HCZ. Also examined was the cytochrome p-450-dependent-oxidative metabolism of (+)-, (-)- and racemic HCZ in vitro using rat liver 9000 x g supernatant fraction. The in vitro metabolism of (-)-HCZ was extremely fast, compared with those of the (+)-isomer and the racemate. The Vmax in vitro showed a good correlation with the CLo in vivo after oral administration (50 mg/kg) of all three forms of HCZ. In vitro study of enantiomeric inhibition of the metabolism showed that (+)-HCZ was a competitive inhibitor of (-)-HCZ metabolism, with a Ki of 6.96 microM. (-)-HCZ was also a competitive inhibitor of (+)-HCZ metabolism, with a Ki of 20.4 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Enantioselective pharmacokinetics of homochlorcyclizine: disposition of (+)- and (-)-homochlorcyclizine after intravenous and oral administration of racemic homochlorcyclizine to rats.

Concentrations of homochlorcyclizine enantiomers in blood, urine, and tissues of the liver, lung, kidney, brain, heart, spleen, intestine and stomach of rats after drug administration were determined by high-performance liquid chromatography on a chiral stationary phase. After intravenous administration (10 mg kg-1), homochlorcyclizine was rapidly distributed in many tissues, with the highest concentration in lung. No differences were found between enantiomers in blood concentrations. After oral administration (50 mg kg-1), the concentrations of the (+)-isomer in nearly all tissues were higher than those of the (-)-isomer. The AUC0-infinity values of the (+)- and (-)-isomers differed significantly. The absorption of racemic homochlorcyclizine from rat small intestine was not enantioselective. These results suggested that the different concentrations between enantiomers after oral administration were not caused by enantioselective absorption or distribution but rather by preferential first-pass metabolism of the (-)-isomer in the liver. The enantioselectivity of metabolism was also demonstrated by in-vitro experiments.

[Synthesis and combined H1-/H2 antagonist activity of mepyramine, pheniramine and cyclizine derivatives with cyanoguanidine, urea and nitroethenediamine partial structures]. / Synthese und kombinierte H1-/H2-antagonistische Aktivität von Mepyramin-, Pheniramin- und Cyclizin-Derivaten mit Cyanoguanidin-, Harnstoff- und Nitroethendiamin-Partialstrukturen.

Compounds with combined histamine H1- and H2-receptor antagonist activity were synthesized by connecting H1- and H2-receptor substructures via cyanoguanidine, urea, or nitroethenediamine moieties. Loss of the strongly basic side-chain nitrogen results in a decrease of H1-receptor activity compared to single reference compounds. At the guinea-pig right atrium (H2-receptor model) compounds with mepyramine or cyclizine structure are also less active than the single references tiotidine, ranitidine, or lamtidine. Nevertheless substances with a pheniramine like partial structure proved to be potent histamine H2-receptor antagonists at the atrium model (about 27 times more active than cimetidine).

Enantioselective pharmacokinetics of homochlorcyclizine. III. Simultaneous determination of (+)- and (-)- homochlorcyclizine in human urine by high-performance liquid chromatography.

A method is described for the simultaneous determination of (+)- and (-)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol-0.02 M acetate buffer (pH 4.7) (25:75,v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (-)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after beta-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (-)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.

Method for optical resolution of racemic homochlorcyclizine and comparison of optical isomers in antihistamine activity and pharmacokinetics.

A method was developed for semi-preparative scale enantioseparation of racemic homochlorcyclizine (HCZ) by high performance liquid chromatography (HPLC) on Chiralcel OD column. The best resolution was achieved using an eluent composed of n-hexane plus 0.2 M isopropylamine. By this method, about 5.0 mg of racemic HCZ could be resolved completely in one run. The optical purity of the enantiomers were both greater than 99.9%. The studies of antihistamine activity on guinea pig ileum demonstrated that l-HCZ is significantly more potent than d- and racemic HCZ. The pharmacokinetics of d- and l-HCZ after oral administration to rats also differed. The successful resolution of racemic HCZ permits comparison of the pharmacokinetics and antihistamine activity of the enantiomers.

Enantioselective pharmacokinetics of homochlorcyclizine. II: Disposition and metabolism of (+)-, (-)- and racemic homochlorcyclizine after oral administration to man.

The pharmacokinetics of a single oral dose of 20 mg (+)-, (-)- and racemic homochlorcyclizine (HCZ) have been studied in humans. The formation of the quarternary ammonium-linked glucuronide was an important metabolic pathway, and the metabolic process was enantioselective as a result of differing urinary excretion rates of (+)-, (-)- and racemic glucuronide. There were significant differences between (+)-, (-)- and racemic HCZ in AUC (0-14 h) and plasma protein binding, but all HCZ enantiomers were slowly absorbed and eliminated (elimination half-lives about 11 h). The results shows help to establish a more efficient dosage regimen for HCZ therapy.

Blood and urine concentrations of cyclizine by nitrogen-phosphorus gas-liquid chromatography.

Gas-liquid chromatography with nitrogen-phosphorus detection was used to analyze blood and urine from a volunteer who ingested 50 mg of cyclizine hydrochloride. A peak blood cyclizine concentration of 69 ng/mL was observed 2 hr after drug administration, and the levels declined thereafter in a biphasic manner, with estimated half-lives of 7 and 24 hr for the early and late phases, respectively. The peak urine cyclizine concentration of 12.5 ng/mL was achieved at 4 hr after administration; only 0.01% of the dose was excreted in the 24 hr urine. Norcyclizine was not observed in blood or urine; however, the detectability of the method for this metabolite is poor relative to the parent drug.