The effect of substituted phenothiazines on the mutagenicity of benzo[a]pyrene

The effect of phenothiazine and 11 of its derivatives on the mutagenicity of benzo[a]pyrene, as measured by the Ames test was investigated. Significant anti-mutagenic activity was detected for 10 phenothiazine derivatives, with the 2-chloro derivative being the most effective inhibitor tested and promazine the only phenothiazine drug tested which has no demonstrable inhibitory activity. It is considered that the anti-mutagenic activity and therefore potentially anticarcinogenic activity of these derivatives should be of interest to epidemiologists.     

Inhibition of mouse embryonic development by calmodulin antagonists

Two-cell mouse embryos were incubated in the presence of calmodulin inhibitors to determine their effect on embryonic development to the blastocyst stage. Calmodulin, a Ca²⁺-dependent regulatory protein, has been localized in the cytoplasm and has been implicated in regulation of many cellular events, such as mitosis. Several concentrations of either commercial or synthesized calmodulin inhibitors were tested. Several phenothiazine sulfoxide derivatives were more effective than the three naphthalene sulonic acid derivatives tested; 2-chloro-10-aminopropyl phenothiazine and 10-aminopropyl phenothiazine were the most potent phenothiazines to inhibit embryonic development at the two-cell stage. The interesting aspect of this study is that phenothiazine sulfoxide derivatives are not potent inhibitors of calmodulin, however, they were successful in inhibiting embryonic development. Potent inhibitors of calmodulin apparently did not penetrate the embryonic membranes because they had no effect.     

Nicotinic Acid as Adjuvant Therapy in Newly Admitted Schizophrenic Patients

A placebo-controlled, comparative clinical study was conducted to test the hypothesis that nicotinic acid as an adjuvant medication has a beneficial therapeutic effect over and above the effect which can be achieved by the administration of phenothiazine drugs alone, over a six-month period, in newly (recently) admitted schizophrenic patients.The most important single finding was that no statistically significant therapeutic difference was seen between the active treatment and the placebo groups; i.e., the addition of nicotinic acid or nicotinamide to the regular phenothiazine treatment regimen did not have any measurable therapeutic effect in this sample of patients. It was shown that patients in the placebo group received a lower total daily amount of phenothiazine drugs than those on either of the active substances. Furthermore, it was noted that the addition of the active substances did not reduce the number of days of hospitalization.     

Photoreduction of membrane bound cytochrome C by excited-state phenothiazine.

Ferricytochrome $\text{c}$ can be reduced in a photochemical reaction by excited state phenothiazine. This reaction is observed between phenothiazine which is solubilized by phospholipid artificial membranes and cytochrome $\text{c}$ which is adsorbed to the membrane surface. Under conditions when cytochrome $\text{c}$ is not bound to the phospholipid, the rate of reduction by phenothiazine is greatly reduced. The phosphorescence of phenothiazine is quenched in the presence of cytochrome $\text{c}$, implying that the excited triplet state interacts with cytochrome $\text{c}$. Oxygen inhibits the reaction since possibly, as a paramagnetic species, it increases intersystem crossing of the excited states of phenothiazine. On the basis of molecular models the proximity between the iron of ferricytochrome $\text{c}$ and phenothiazine is estimated to be over 20 Å.     

Neither lipophilicity nor membrane-perturbing potency of phenothiazine maleates correlate with the ability to inhibit P-glycoprotein transport activity

Although phenothiazines are known as multidrug resistance modifiers, the molecular mechanism of their activity remains unclear. Since phenothiazine molecules are amphiphilic, the interactions with membrane lipids may be related, at least partially, to their biological effects. Using the set of phenothiazine maleates differing in the type of phenothiazine ring substitution at position 2 and/or in the length of the alkyl bridge-connecting ring system and side chain group, we investigated if their ability to modulate the multidrug resistance of cancer cells correlated with model membrane perturbing potency. The influence exerted on lipid bilayers was determined by liposome/buffer partition coefficient measurements (using the absorption spectra second-derivative method), fluorescence spectroscopy and calorimetry. Biological effects were assessed by a flow cytometric functional test based on differential accumulation of fluorescent probe DiOC(2)(3) by parental and drug-resistant cells. We found that all phenothiazine maleates were incorporated into lipid bilayers and altered their biophysical properties. With only few exceptions, the extent of membrane perturbation induced by phenothiazine maleates correlated with their lipophilicity. Within the group of studied derivatives, the compounds substituted with CF(3)- at position 2 of phenothiazine ring were the most active membrane perturbants. No clear relation was found between effects exerted by phenothiazine maleates on model membranes and their ability to modulate P-glycoprotein transport activity.     

New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potency

The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules. Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix. In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H-, Cl-, CF(3)-) and in the side chain group (NHCO(2)CH(3) or NHSO(2)CH(3)). Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (logP) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers. It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group.     

Calcium ion dependent covalent modification of calmodulin with norchlorpromazine isothiocyanate

Calmodulin forms a covalent, one to one, complex with 3H-labeled norchlorpromazine isothiocyanate. Complex formation was monitored by high-performance liquid chromatography using a CN reverse-phase column which resolves calmodulin, the calmodulin-norchlorpromazine adduct, and norchlorpromazine isothiocyanate. Formation of the adduct requires Ca2+ and is not observed with norchlorpromazine. The one to one calmodulin-norchlorpromazine complex does not activate phosphodiesterase but can interact with the enzyme and competitively inhibit its stimulation by calmodulin. High concentrations of trifluoperazine inhibit whereas low concentrations stimulate complex formation. This apparent potentiation of the interaction of calmodulin with norchlorpromazine by another phenothiazine suggests that calmodulin contains at least two phenothiazine binding sites and that the binding of phenothiazine to calmodulin is cooperative.     

Phenothiazine Poisoning A Review of 48 Cases

Of 48 cases of phenothiazine poisoning that were analyzed, 34 were attributed to suicide attempts, nine to accidental ingestion, and five to drug reactions.As outpatient treatment of schizophrenia increases, cases of over-dose with phenothiazine drugs may be expected to increase also.The prescribing of multiple phenothiazines and antidepressants is probably contributory to the occurrence of mixed drug ingestions.The symptoms and signs of phenothiazine poisoning are largely predictable if the atropine-like, alpha-blocking, quinidine-like, and extrapyramidal actions of phenothiazines are appreciated. Unexplainable tachypnea and paradoxical miosis were noted in severe cases.In one case in the study phenothiazine intoxication was present in the newborn infant of a schizophrenic mother.     

Peroxidase catalysed formation of cytotoxic prooxidant phenothiazine free radicals at physiological pH

The antipsychotic phenothiazines may have other therapeutic applications because of their ability to kill bacteria, plasmids and tumor cells. They are also known to undergo a peroxidase-catalysed oxidation to form cation radicals that are stable at acid pH, but are not detected at a neutral pH. The objective of this project was to determine whether phenothiazine cation radical metabolites could cause oxidative stress at a neutral pH resulting in cytotoxicity. At a neutral pH, catalytic amounts of phenothiazines were found to be oxidised by a peroxidase/H2O2 system and also caused ascorbate, GSH and NADH cooxidation. NADH and GSH co-oxidation was accompanied by oxygen uptake and was increased by the addition of catalytic amounts of superoxide dismutase, indicating that the superoxide radical was formed. The phenothazines were different from other peroxidase substrates in that the NADH, ascorbate or GSH cooxidation was faster at pH 6.0 than pH 7.4, thereby partly reflecting the cation radical stability. The order of catalytic effectiveness found was promazine > chlorpromazine > trifluoperazine. Peroxidase/H2O2 also markedly increased phenothiazine cytotoxicity towards isolated rat hepatocytes at nontoxic phenothiazine concentrations. At both pH 6.0 and 7.4, the same order of phenothiazine catalytic effectiveness was observed as seen in the co-oxidation experiments. Cytotoxicity to hepatocytes could be attributed to oxidative stress as most hepatocyte glutathione oxidation and lipid peroxidation preceded phenothiazine induced cytotoxicity and that cytotoxicity was prevented by the antioxidant butylated hydroxyanisole. This hepatocyte/peroxidase/H2O2 system could be a useful model for studying drug induced idiosyncratic hepatic injury enhanced by inflammation.     

Purification of recombinant proteins based on the interaction between a phenothiazine-derivatized column and a calmodulin fusion tail.

A method to purify proteins by fusing them to the Ca2+-dependent protein calmodulin is described by using glutathione-S-transferase (GST) from Schistosoma japonicum as a model. Glutathione-S-transferase was genetically fused to calmodulin (CaM). The designed GST-CaM fusion protein has a selective factor Xa cleavage site located between the C-terminus of GST and the N-terminus of CaM. The recombinant fusion protein was expressed in Escherichia coli, and the crude cell extract was loaded onto a phenothiazine affinity column in the presence of Ca2+. Calmodulin was used as an affinity tail to enable binding of the fusion protein to the phenothiazine column. Removal of Ca2+ with a calcium-complexing solution causes elution of the fusion protein. The GST-CaM fusion protein was then digested with factor Xa, and the target protein GST was isolated. The purity of the isolated GST was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

Broad specific enzyme-linked immunosorbent assay for determination of residual phenothiazine drugs in swine tissues

In this study, a novel generic hapten of phenothiazine drugs was synthesized by derivatization of 2-chlorophenothiazine with sodium bromoacetate. Then the hapten was coupled to bovine serum albumin for production of the monoclonal antibody. Results showed that the obtained monoclonal antibody recognized five phenothiazine drugs simultaneously: chlorpromazine, promethazine, acepromazine, perphenazine, and fluphenazine. After evaluation of different coating antigens, a heterologous competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of the five phenothiazine drugs in swine tissues (muscle, liver, and kidney). The cross-reactivities to the five analytes were in the range of 71 to 98%, and the limits of detection were in the range of 0.2 to 0.4 ng/ml, depending on the drug. Their recoveries from the fortified blank samples were in the range of 73.8 to 96.2%, with coefficients of variation in the range of 4.1 to 14.3%. This is the first study reporting a broad specific immunoassay for multi-determination of the residues of five phenothiazine drugs in animal-derived foods.     

Peptide chemistry applied to a new family of phenothiazine-containing inhibitors of human farnesyltransferase

Novel phenothiazine derivatives bearing an amino acid residue were synthesized via peptide chemistry, and evaluated for their inhibitory potential on human farnesyltransferase. The phenothiazine unit proved to be an important bulky unit in the structure of the synthesized inhibitors. Propargyl ester 20 bearing a tyrosine residue exhibited the best biological potential in vitro in the present study. Further syntheses and biological evaluation of phenothiazine derivatives are necessary in order to gain a full view of SAR in this family of farnesyltransferase inhibitors.     

EVIDENCE THAT METHADONE BLOCKS DOPAMINE RECEPTORS IN THE BRAIN

Abstract— This study has shown that methadone shares with phenothiazine and butyro-phenoneneuroleptics several pharmacological and biochemical actions:thus, D,L-methadone causes catalepsy and hypothermia, blocks apomorphine-induced gnawing, increases brain homovanillic acid levels and stimulates brain dopamine synthesis. The dextro isomer of methadone is inactive. α-Methyl-tyrosine potentiates and apomorphine reverses methadone-induced catalepsy. The data suggest that methadone, like butyrophenone and phenothiazine neuroleptics, blocks dopamine receptors in brain.     

Hollow fiber-liquid phase microextraction combined with gas chromatography for the determination of phenothiazine drugs in urine

A simple method of hollow fiber-liquid phase microextraction (HF-LPME) combined with gas chromatography (GC) was developed for the analysis of four phenothiazine drugs (promethazine, promazine, chlorpromazine and trifluoperazine) in human urine samples. All variables affecting the extraction of target analytes including organic solvent type, stirring rate, extraction time, extraction temperature, pH of sample solution and ionic strength were carefully studied and optimized. Under the optimal conditions, the analytical performance of HF-LPME-GC-flame photometric detector (FPD) and HF-LPME-GC-flame ionization detector (FID) were evaluated and compared. The results showed that the HF-LPME-GC-FID was more sensitive than HF-LPME-GC-FPD for the determination of four target phenothiazine drugs, while the signal peak shape and resolution obtained by HF-LPME-GC-FPD was better than that obtained by HF-LPME-GC-FID. HF-LPME-GC-FPD/FID was successfully applied for the assay of the interested phenothiazine drugs in urine sample, and the excretion of the drugs was also investigated by monitoring the variation of the concentration of chlorpromazine in urine of a psychopath within 8 h after drug-taking. The proposed method provided an effective and fast way for the therapeutic drug monitoring (TDM) of phenothiazine.     

Thiopropazate Hydrochloride in Persistent Dyskinesia

Thiopropazate (Dartalan) was found to be significantly more effective than a placebo in relieving dyskinesia in 23 patients with functional psychosis and persistent dyskinesia associated with prolonged phenothiazine therapy. Each patient whose dyskinesia had persisted unchanged for at least one month after phenothiazine withdrawal received thiopropazate by mouth for three weeks and the placebo for a similar period. Patients were evaluated before the trial, at three weeks, and at six weeks.The drug also improved psychotic behaviour. Possible side effects, which were generally mild, were noted in eight patients, of whom six had Parkinsonism and four drowsiness. None had side effects while on the placebo.The findings indicate that thiopropazate is of value in persistent dyskinesia associated with prolonged phenothiazine intake—a condition hitherto unresponsive to other treatment. Further research is required to determine the long-term effectiveness of the drug.     

THIORIDAZINE (MELLARIL?) IN PSYCHIATRIC PATIENTS

Thioridazine (Mellaril®) was given to 104 psychiatric patients with a variety of illnesses, chiefly schizophrenic reactions. Of 14 patients treated in a double-blind study with successive one-month courses of drug or placebo, nine improved most on the drug and only one on placebo. These results, although limited, confirm a definite therapeutic action for this compound.Nine of 24 patients were significantly improved after treatment with thioridazine for an average of four months following previous treatwith other phenothiazine tranquilizers. Of ten patients treated intensively with thioridazine after they had not responded to other phenothiazine drugs, two were definitely improved and three were slightly improved. Twenty-eight of 56 patients treated from the outset with thioridazine were significantly improved after an average of six months. Most patients received from 100 to 400 mg. daily. These results were comparable to those obtained from other potent phenothiazine tranquilizers. The drug is particularly advantageous for a group of schizophrenic patients who are sometimes made worse by other phenothiazine derivatives or rauwolfia alkaloids. It should also be suitable for treating patients with psychoneuroses and chronic brain syndromes.Only minimal side reactions were observed, chiefly drowsiness, dizziness and nasal stuffiness. Weight gain occurred frequently during treatment.     

Phenothiazine‐Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin‐Mediated Endocytosis

Chlorpromazine is a phenothiazine‐derived antipsychotic drug (APD) that inhibits clathrin‐mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine‐derived APDs inhibited dynamin I (dynI) in the 2–12 µm range, the most potent being trifluoperazine (IC₅₀ 2.6 ± 0.7 µm ). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8‐ to 10‐fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine‐derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine‐derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin‐active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure–activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP‐2 recruitment in the formation of clathrin‐coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP‐2 interaction with amphiphysin or clathrin. Super‐resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP‐2, nor AP‐2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine‐derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in‐cell target in endocytosis.      

The synthesis and reaction of a specific affinity label for the hydrophobic drug-binding domains of calmodulin

An affinity-labeling reagent for the two hydrophobic drug-binding domains of calmodulin has been prepared and its reaction with calmodulin characterized. The reagent, 10-(3-propionyloxysuccinimide)-2-(trifluoromethyl)phenothiazine, was shown to be very specific labeling reagent for these domains. Its specificity was demonstrated by the following observations. 1) Previous reports have shown that Ca2+ is required for phenothiazine binding to calmodulin, and here we show that the affinity-labeling reagent reacts with and inactivates calmodulin in the presence of Ca2+, but not in its absence. 2) Inclusion of trifluoperazine, fluphenazine, W-7, or 10-(3-aminopropyl)-2-(trifluoromethyl)phenothiazine in the reaction mixture protected calmodulin from inactivation by the reagent. 3) Inactivation by the reagent yielded calmodulin that was no longer retained on a phenothiazine-Sepharose column under conditions in which unreacted calmodulin was retained. 4) The measured stoichiometry of the reaction in the presence of excess reagent was 2.1 mol of reagent per mol of calmodulin which agrees well with previous reports of two high-affinity phenothiazine-binding sites on calmodulin. 5) The stoichiometry of the reaction was further confirmed by tryptic peptide maps which show two phenothiazine-labeled peptides unique to the fully reacted protein. 6) The spectral properties of the reagent, while attached to calmodulin, change in the presence of Ca2+ in a manner consistent with the known effects of Ca2+ binding by calmodulin on these hydrophobic domains. The specificity of the reagent makes it useful for further characterization of these hydrophobic binding domains on calmodulin.     

Adenylate cyclase activity in cyanobacteria: activation by Ca(2+)-calmodulin and a calmodulin-like activity

An adenylate cyclase activity was partially characterized in the cyanobacterium Anabaena sp. The enzyme activity is found in soluble cell fractions and shows an apparent molecular weight of about 183,400. This adenylate cyclase is activated by Ca2+ and bovine brain or spinach calmodulin and it is inhibited by EGTA and some phenothiazine derivatives. Furthermore, Anabaena sp. extracts contain a calmodulin-like activity which stimulates bovine brain cyclic AMP phosphodiesterase and the Anabaena adenylate cyclase. EGTA and phenothiazine derivatives block the cyanobacterial modulator effect.     

Phenotypes ofdnaA mutants ofEscherichia coli sensitive to phenothiazine derivatives

The activation of DnaA protein by cardiolipin is inhibited by fluphenazinein vitro. We therefore examined the sensitivity of temperature-sensitivednaA mutants ofEscherichia coli to fluphenazine and other phenothiazine derivatives. Among the eightdnaA mutants tested,dnaA5, dnaA46 dnaA602, anddnaA604, mutants with mutations in the putative ATP binding site of DnaA protein, showed higher sensitivities to phenothiazine derivatives than did the wild-type strain. ThednaA508 anddnaA167 mutants, which have mutations in the N-terminal region of DnaA protein, also showed higher sensitivities to phenothiazine derivatives. On the other hand, thednaA204 anddnaA205 mutants, with lesions in the C-terminal region of the DnaA protein, showed the same sensitivity to phenothiazine derivatives as the wild-type strain. Complementation analysis with a plasmid containing the wild-typednaA gene and phage P1-mediated transduction confirmed thatdnaA mutations are responsible for these sensitivity phenotypes.