Synthesis and anti-inflammatory effects of new piperazine and ethanolamine derivatives of H(1)-antihistaminic drugs.

In addition to their antihistamine effects, H1-receptor antagonists possess pharmacological properties that are not uniformly distributed among this class of drugs, such as anti-inflammatory, anti-allergic and antiplatelet activities. In this paper, Cyclizine (1-benzhydryl-4-methyl-piperazine, I), bromodiphenhydramine (2-[(4-bromophenyl)-phenylmethoxy]-N, N-dimethylethanamine, II) and some of their new piperazine and ethanolamine derivatives (III-VIII) inducing changes in substitution of phenyl and amine moieties were synthesized and their acute and chronic antiinflammatory effects were evaluated by standard pharmacological tests. The results showed that substitution of phenyl by tolyl, anisol and cumene groups in piperazine family could remarkably decrease acute inflammation in these new drugs. Also, substitution of dimethylamine by morpholine group could not decrease this inflammation in new synthesized ethanolamine family. But the results from the cotton pellet-induced granuloma formation in rats showed that none of drugs (I-VIII) were effective to reduce the chronic inflammation.

Poisonings with diphenhydramine--a survey of 68 clinical and 55 death cases.

The antihistaminic drug diphenhydramine (DPH) is mainly used as a sedative, hypnotic and antiemetic. In many countries it is over-the-counter available, very common, and generally regarded as a harmless drug. Sixty-eight non-fatal and 55 fatal poisonings with DPH alone or in combination with other drugs were investigated in the Institute of Legal Medicine of the University Hospital Charité between 1992 and 2004. The analytical investigations were performed by HPLC with photodiode array detector (HPLC-DAD). The DPH concentrations ranged from 0.5 to 8.9 microg/mL in the non-fatal cases and from 0.3 to 119 microg/mL in fatal cases. The intoxication symptoms stated during emergency admission were inconsistent, with somnolence, sedation and retardation on one hand and tachycardia, anticholinergic syndrome, agitation, hallucinations, confusion, tremor, convulsions, delirium and coma on the other. In three cases rhabdomyolysis occurred. A concentration above 5 microg/mL can be regarded as potentially lethal. In many of the survivors the time course of the concentrations of DPH and the metabolites desmethyldiphenhydramine (DM-DPH) and diphenylmethoxyacetic acid (DPMA) were investigated. Whereas DM-DPH is present in blood from the very beginning because of the high first pass metabolism, DPMA is slowly formed over several metabolic steps. For this reason, the concentration ratio DPMA/DPH can be used for an approximate estimation of the time between drug intake and sampling in clinical cases or of the survival time after drug ingestion in death cases. In some of the deaths the concentrations in heart blood were much higher than in venous blood. This is explained mainly by agonal aspiration of the vomited gastric content. Besides the majority of suicidal cases also a case of child maltreatment and a case, in which the drug was forcibly administrated in a drug facilitated crime, were investigated. From the results it follows that diphenhydramine is not less poisonous than other prescribed hypnotics. However, despite the hallucinogenic effects, an abuse for recreational purposes was not observed until now.

Cationic amphiphilic drugs and platelet phospholipase A(2) (cPLA(2)).

The aim of the study was to verify and compare the effect of cationic amphiphilic drugs (CAD) from different pharmacological groups on activation of platelet phospholipase A2 (PLA2)--the essential enzyme of arachidonic pathway in blood platelets. Beta-adrenoceptor-blocking (BAB) drugs inhibited platelet aggregation in the rank order of potency: propranolol>alprenolol>metipranolol>atenolol. The higher the inhibition of arachidonic acid (AA) liberation by BAB drugs, the higher the inhibition of aggregation. Similarly did the H1-histamine antagonists bromadryl (BRO) and dithiaden (DIT) as well as the antimalarial chloroquine (CQ) show antiplatelet effect in vitro in the rank order of potency: DIT>BRO>CQ. Dose-dependent inhibition of aggregation was followed by the inhibition of AA liberation from membrane phospholipids of platelets stimulated either at the receptor site (thrombin) or by a stimulus bypassing membrane receptors (Ca2+ ionophore A23187). The rank order potency for inhibition of stimulated 3H-AA liberation from membrane phospholipids was: (a) for BAB drugs: propranolol>alprenolol>metipranolol, (b) for other drugs: DIT>BRO>CQ. The investigated drugs' interference with stimulated liberation of AA showed nonspecific inhibition of platelet cytosolic PLA2 (cPLA2) by these drugs at intracellular level. The results revealed that besides the inhibition of cyclooxygenase pathway and receptors for adenosine diphosphate (ADP) and glycoproteins Gp IIbIIIa, the interaction of drugs with cPLA2 may represent a further site for antiplatelet action.

Titrimetric and spectrophotometric assay of some antihistamines through the determination of the chloride of their hydrochlorides.

Two simple, rapid and reliable methods for the determination of four antihistamines based on the measurement of the chloride of their hydrochlorides are described. In the titrimetric method, the chloride content of each drug is determined by titrating with mercury(II) nitrate using diphenylcarbazone-bromothymol blue as indicator. In the spectrophotometric method, to a fixed concentration of mercury(II)-diphenylcarbazone complex different amounts of drug are added and the decrease in absorbance of mercury(II)-diphenylcarbazone complex, consequent to the replacement of diphenylcarbazone of the complex by the chloride of the drug, was measured at 540 nm. The stoichiometry of the reaction that forms the basis for titrimetry is assessed. Different variables affecting the color formation in spectrophotometry were studied and optimized. At the wavelength of maximum absorption, Beer's law is obeyed in the 0-100 microg ml(-1) range. The molar absorptivity and Sandell sensitivity are calculated. The proposed methods were applied for the analysis of pharmaceutical formulations containing these drugs. Statistical treatment of the experimental results indicates that the procedures are precise and accurate. Excipients used as additives in pharmaceutical formulations did not interfere in the proposed procedures as shown by the recovery studies.

Vitrification of mouse oocytes using a nylon loop.

Cryopreservation of mouse oocytes was improved by the use of ultra-rapid vitrification using a nylon loop of 0.5 mm diameter. Oocytes that were vitrified using the loop survived at high rates and were fertilized following a small hole being made in the zona pellucida (69.8%) and developed to the blastocyst stage in culture (67.4%) at similar rates to that of oocytes that were not cryopreserved. Blastocysts resulting from oocytes vitrified using the nylon loop had similar development of the inner cell mass and trophectoderm as blastocysts from non-cryopreserved oocytes. In contrast, oocytes that were cryopreserved using a slow-freezing protocol where most of the Na+ is replaced with choline had lower rates of fertilization (39.5%), reduced development to the blastocyst stage (25.7%), and blastocysts had reduced development of the inner cell mass. Blastocysts derived from oocytes that were vitrified with the nylon loop were able to implant (88.0%) and develop into fetuses (56.5%) at significantly higher rates compared to blastocysts derived from oocytes that were slow-frozen (52.4 and 26.2%, respectively). Vitrification of mouse oocytes using the nylon loop results in the retention of viability of the oocytes and subsequent embryos.

Pharmacological intervention with platelet phospholipase A2.

BACKGROUND: Metabolites of arachidonic acid are important regulatory substances in blood platelets. They participate in platelet adhesion and aggregation, and pharmacological intervention with arachidonate cascade is widely used in therapy of hyperactive platelets and in the prevention of thromboembolic complications. AIM OF THE STUDY: To verify and compare the effect of cationic amphiphilic drugs (CAD) from different pharmacological groups on activation of platelet phospholipase A2--the essential enzyme of arachidonic pathway in blood platelets. METHODS: Blood platelets were isolated from human and rat blood by differential centrifugation and aggregation was measured in pretreated and subsequently stimulated platelets in a dual channel aggregometer and recorded on a linear recorder. Activity of cytosolic phospholipase A2 (cPLA2) was determined by means of arachidonic acid liberation from platelet membrane phospholipids incorporated as triciated radionuclide. The radioactivity was determined by liquid scintillation method in Packard TriCarb 2500T. RESULTS: Cationic amphiphilic drugs of the beta-adrenoceptor blocking group inhibited platelet aggregation in the rank order of potency: propranolol > alprenolol > metipranolol > atenolol. Similarly did the H1-histamine antagonists bromadryl and dithiaden as well as the antimalarial chloroquine show antiplatelet effect in vitro in the rank order of potency: dithiaden > or = bromadryl > or = chloroquine. Dose-dependent inhibition of aggregation was followed by inhibition of arachidonic acid liberation from membrane phospholipids of platelets stimulated at receptor site (thrombin) or by a stimulus bypassing membrane receptors (Ca2+ ionophore A23187). The rank order potency for inhibition of stimulated 3H-AA liberation from membrane phospholipids was: a) for BAB drugs: propranolol > or = alprenolol > metipranolol, b) for other drugs: dithiaden > bromadryl > chloroquine. CONCLUSION: The drugs investigated interference with liberation of stimulated arachidonic acid showed nonspecific inhibition of platelet cytosolic phospholipase A2 by these drugs at intracellular level. The results revealed that besides inhibition of cyclooxygenase pathway, and of receptors for ADP and glycoproteins Gp IIb/IIIa, interaction of drugs with cPLA2 may represent a further site for antiplatelet action. (Fig. 5, Ref. 39.)

Inhibition of Na(+) current by diphenhydramine and other diphenyl compounds: molecular determinants of selective binding to the inactivated channels.

Diphenhydramine is an H1 histamine receptor antagonist, yet it also has a clinically useful local anesthetic effect. We found that diphenhydramine inhibits the neuronal Na(+) current, and the inhibition is stronger with more positive holding potentials. The dissociation constant between diphenhydramine and the inactivated Na(+) channel is approximately 10 microM, whereas the dissociation constant between diphenhydramine and the resting channel is more than 300 microM. The local anesthetic effect of diphenhydramine thus is ascribable to inhibition of Na(+) current by selective binding of the drug to the inactivated channels. Most interestingly, many other compounds, such as the anti-inflammatory drug diclofenac, the anticonvulsant drug phenytoin, the antidepressant drug imipramine, and the anticholinergic drug benztropine, have similar effects on neuronal Na(+) current. There is no apparent common motif in the chemical structure of these compounds, except that they all contain two phenyl groups. Molecular modeling further shows that the two benzene rings in all these drugs have very similar spatial orientations (stem bond angle, approximately 110 degrees; center-center distance, approximately 5 A). In contrast, the two phenyl groups in phenylbutazone, a drug that has only a slight effect on Na(+) current, are oriented in quite a different way. These findings strongly suggest that the two phenyl groups are the key ligands interacting with the channel. Because the binding counterpart of a benzene ring usually is also a benzene ring, some aromatic side chain groups of the Na(+) channel presumably are realigned during the gating process to make the very different affinity to the aforementioned drugs between the inactivated and the resting channels.

Simultaneous determination of diphenhydramine, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine using liquid chromatography/electrospray tandem mass spectrometry.

Our studies on drug disposition in chronically instrumented pregnant sheep involve simultaneous administration of the antihistamine diphenhydramine (DPHM), its deuterated analogue ([2H10]DPHM) and their metabolites to the mother or the fetus via various routes. Such studies require sensitive and selective mass spectrometric methods for quantitation of these labeled and unlabeled compounds in order to assess comparative maternal and fetal drug metabolism. The objective of this study was to develop and validate a liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantitation of DPHM, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine. Samples spiked with the analytes and the internal standard, orphenadrine, were processed using liquid-liquid extraction. The extract was chromatographed on a propylamino LC column and MS/MS detection was performed in the positive ion electrospray mode using multiple reaction monitoring. The linear concentration ranges of the calibration curves for the N-oxides and the parent amines were 0.4-100.0 and 0.2-250.0 ng ml-1, respectively. In validation tests, the assay exhibited acceptable variability (< or = 15% at analyte concentrations below 2.0 ng ml-1 and < 10% at all other concentrations) and bias (< 15% at all concentrations), and the analytes were stable under a variety of sample handling conditions. Using this method, the labeled and unlabeled N-oxide metabolite was identified in fetal plasma after DPHM and [2H10]DPHM administration. This method will be used further to examine the comparative metabolism of diphenhydramine to its N-oxide metabolite in the mother and the fetus.

Inhibition of blood platelet functions by cationic amphiphilic drugs in relation to their physico-chemical properties.

The effects of bromadryl, dithiaden, chloroquine and propranolol on thrombin-stimulated rat platelet aggregation (measured turbidimetrically) and thromboxane B2 generation (detected by an RIA method) were compared with four selected physico-chemical parameters of these drugs. Platelet aggregation was inhibited in the rank order of potency: bromadryl > dithiaden > propranolol > chloroquine, which corresponded with the decrease in the net charge of the terminal methyl-(or ethyl-) groups in the side chain and with the increase of the dipole moment of drug molecules. On the other hand, the rank order of potency in which the drugs tested inhibited thromboxane B2 formation (chloroquine > dithiaden > bromadryl > propranolol) correlated well with the decline in molar refractivity of the drugs. No relationship was found between inhibitory effects of drugs and their partition coefficients. The results presented indicate that inhibition of platelet functions might consist of several types of drug-cell interactions, depend on the structure and physico-chemical properties of the drugs and cannot be estimated simply on the basis of partition coefficients.

Synthesis and pharmacology of combined histamine H1-/H2-receptor antagonists containing diphenhydramine and cyproheptadine derivatives.

The classical histamine H1-receptor antagonists diphenhydramine (3a) and cyproheptadine (9) and their derivatives (3b-d, 10) were connected with a 2-guanidinothiazole containing structure (28) derived from the H2-receptor antagonist tiotidine in order to obtain combined H1-/H2-receptor antagonists. The two moieties were not directly linked together, but were separated by a polymethylene spacer and a polar group (nitroethenediamine or urea). Thus 12 compounds were obtained that proved in vitro to possess high H1- and H2-receptor antagonist activity at the isolated guinea-pig ileum (H1) and the isolated guinea-pig right atrium (H2), respectively. The incorporation of the diphenhydramine as well as the cyproheptadine component provides high affinity to H1-receptors. The tricyclic cyproheptadine and its 10,11-dihydro derivative (30-32, 34), however, cause a decrease of H2-receptor antagonist potency compared to the diphenhydramines (29a-d, 33a-d). Using nitroethenediamine as the polar group is apparently more favourable to H1- and H2-receptor affinity as the urea function. All compounds elicit a dual mode of competitive and noncompetitive antagonism. Among the novel compounds the nitroethenediamines with 4-fluoro- or 4-methyl-substituted diphenhydramine as H1-receptor antagonist moiety (29c, d) display the most potent H1- and H2-receptor antagonist effects. The presented concept is a very promising way to combine H1- and H2-receptor antagonist properties in one molecule.

[The effect of antihistamines on stimulated blood platelet functions]. / Ovplyvnenie stimulovaných funkcií krvných dosticiek antihistaminikami.

The histamine receptor H1-antagonists of cationic amphiphilic drug (CAD) structure BromadrylR (BRO) and DithiadenR (DIT) were investigated on stimulated functions of blood platelets in vitro. Both drugs dose-dependently decreased platelet aggregation. BRO significantly decreased aggregation in concentrations of 20 and 200 mumol/l in thrombin and ADP stimulated platelets, respectively. DIT was 10 times less active as compared with BRO. A dose-dependent inhibitory effect of BRO and DIT was demonstrated on thrombin stimulated production of malondialdehyde (MDA) and thromboxane (TXB2) generation in platelets. A significant decrease in MDA production and TXB2 generation was measured with BRO and DIT in 10 mumol/l concentration. Results showed that the antihistaminic drugs BRO and DIT inhibited stimulated aggregation in relation to membrane phospholipid peroxidation and arachidonic cascade activation in platelets. These data, along with results from studies on the effect of other CAD on platelets, revealed that antihistaminic drugs interfere with stimulated aggregation by inhibiting phospholipase A2 rather than the intraplatelet histamine receptor.

Effect of the antihistaminic drug Bromadryl on ex vivo platelet functions during gestation in rats.

The effect of the antihistaminic drug Bromadryl on stimulated platelet aggregation was studied in pregnant rats. Bromadryl was administered orally in doses 1, 5 and 10 mg/kg from day 2 to day 19 of gestation. On day 20 of gestation blood samples were taken and the aggregation of platelets was measured after stimulation with 9 different concentrations of ADP in the concentration range 0.2 to 100 mumol/l. During gestation, platelet aggregation was significantly increased in comparison to non-pregnant controls. This was indicated in pregnant rats by the increased maximum amplitude of the aggregation curve as well as by the decreased mean effective concentration of ADP. The treatment of dams with Bromadryl during pregnancy resulted in decreased platelet aggregation. The dose-dependent inhibitory effect of the drug was more pronounced at higher stimulus concentrations. The presented results confirmed the antiplatelet activity of Bromadryl observed previously in vitro.

Antihistaminic drug bromadryl and stimulated platelet aggregation.

The H1-receptor antagonist bromadryl dose-dependently inhibited stimulated rat platelet aggregation in vitro. Bromadryl was 10 times more effective on the secondary aggregation of thrombin-stimulated platelets compared with its inhibition of ADP-stimulated primary platelet aggregation in plasma. The inhibition of aggregation was accompanied by a dose-dependent inhibition of thrombin-stimulated malondialdehyde formation and thromboxane B2 production. The results indicate that bromadryl may interfere intracellularly with membrane phospholipid peroxidation and the arachidonic acid metabolism of stimulated platelets. Bromadryl, like other cationic amphiphilic drugs, may inhibit stimulated platelet functions by decreasing the stimulus-induced activation of phospholipase A2. Our results support the possible interference of bromadryl with histamine as an intraplatelet messenger responsible for aggregation.

Cytochrome P-450 metabolic-intermediate complex formation with a series of diphenhydramine analogues.

A series of diphenhydramine analogues have been studied with regard to their formation of a metabolic intermediate (MI) during their biotransformation in phenobarbital induced rat hepatic microsomes. The MI forms a complex with reduced cytochrome P-450. MI complexation of cytochrome P-450 may result in drug-drug interactions and/or in cumulation of the parent compound. The extent of MI complex formation could be correlated with the lipophilicity of the substrates in a parabolic manner. A hydrophobic pocket of limited dimensions in cytochrome P-450 for the N-alkyl substituent of the substrates can be assumed. Moreover our data indicate a role for the O-atom in the diphenhydramine analogues for the interaction with cytochrome P-450.

Studies on synergism between penicillins and ambodryl (bromodiphenhydramine HCl), an antihistamine with antimicrobial property.

With respect to 31 different selected test bacteria, all sensitive to benzyl penicillin (Pc), ampicillin (Ap), methicillin (Me), ceporan (Ce), cloxacillin (Cx), streptomycin (Sm), kanamycin (Km), gentamicin (Gm), chloramphenicol (Cm), tetracycline (Tc), polymyxin (Pm) as well as ambodryl [Am; an antihistamine (bromodiphenhydramine HCl) with distinct antimicrobial properties], it was found that Am in combination either with Pc, Ap, Ce or Me consistently showed enhancement of antimicrobial effects resulting from synergism. A combination of Am with either Sm, Km, Gm or Tc, on the other hand, showed only additive effects. An interaction of the activities of Am with Pm also resulted in indifference effects. Determination of the area of inhibition zone, calculated from its diameter for the degree of synergism in case of Am and Pc, showed these synergistic effects to be significant (P less than 0.05) in comparison with their individual effects: this was corroborated by the determination of the fractional inhibitory concentration (FIC) index which was found to be less than 0.5. The synergism of Am-Pc combination was confirmed by in vivo studies by challenging mice with a virulent strain of S. typhimurium and looking for protection.

Structural features of some diphenhydramine analogues that determine the interaction with rat liver cytochrome P-450.

The aim of this study was to define the structural characteristics in a series of 21 analogues of the anti-histaminergic drug diphenhydramine which are important for the interaction with cytochrome P-450. The compounds gave substrate (type I) binding spectra with rat hepatic microsomal cytochrome P-450. The main findings were: (1) two phenyl rings are needed for strong binding: saturation or elimination of one ring, or restriction of two phenyls with a two-carbon bridge results in a decrease of binding, (2) substitution on one or both aromatic rings has only a small influence on binding, (3) an amine nitrogen contributes to better binding; decrease or absence of basicity weakens binding, and (4) a chain of 4 to 7 atoms connecting the basic centre with the aromatic part is needed; reduction of the chain length, or restriction of it to a cyclic structure causes decrease or loss of binding ability.

Pharmacokinetics of diphenhydramine and a demethylated metabolite following intravenous and oral administration.

Ten healthy volunteers received a single 50-mg dose of diphenhydramine (DP) hydrochloride intravenously and orally on two separate occasions. Kinetics of DP and a major demethylated metabolite (DMDP) were determined from multiple plasma samples drawn during a 24- to 48-hour period after dosage. Modification of a gas chromatographic (GC) technique allowed simultaneous quantitation of DP and DMDP. Mean kinetic variables for DP after intravenous (IV) dosage were: volume of distribution, 4.5 L/kg; elimination half-life, 8.4 hours; clearance, 6.2 mL/min/kg. After oral DP administration, a peak plasma level of 66 ng/mL was reached 2.3 hours after dosage. Systemic availability was 72%, nearly identical to the predicted estimate (71%) based on clearance of IV DP relative to hepatic blood flow. Appearance of the metabolite, DMDP, mirrored disappearance of DP; the area under the plasma concentration-time curve (AUC) for DMDP was highly correlated (r = .79, P less than .05) with a clearance of IV DP. However, metabolite AUC was significantly higher after oral as opposed to IV DP (218 vs 145 hr-ng/mL, P less than .05). Because DP and DMDP elute nearly identically on standard GC systems, methodologic modifications are needed to resolve them. Coelution of the two compounds could bias kinetic data based on plasma concentration presumed to be specific for intact DP.