Treatment of SEC62 over-expressing tumors by Thapsigargin and Trifluoperazine.

Treatment with analogues of the SERCA-inhibitor Thapsigargin is a promising new approach for a wide variety of cancer entities. However, our previous studies on various tumor cells suggested resistance of SEC62 over-expressing tumors to this treatment. Therefore, we proposed the novel concept that e.g. lung-, prostate-, and thyroid-cancer patients should be tested for SEC62 over-expression, and developed a novel therapeutic strategy for a combinatorial treatment of SEC62 over-expressing tumors. The latter was based on the observations that treatment of SEC62 over-expressing tumor cells with SEC62-targeting siRNAs showed less resistance to Thapsigargin as well as a reduction in migratory potential and that the siRNA effects can be mimicked by the Calmodulin antagonist Trifluoperazine. Therefore, the combinatorial treatment of SEC62 over-expressing tumors was proposed to involve Thapsigargin and Trifluoperazine. Here, we addressed the impact of Thapsigargin and Trifluoperazine in separate and combined treatments of heterotopic tumors, induced by inoculation of human hypopharyngeal squamous cell carcinoma (FaDu)-cells into the mouse flank. Seeding of the tumor cells and/or their growth rate were significantly reduced by all three treatments, suggesting Trifluoperazine is a small molecule to be considered for future therapeutic strategies for patients, suffering from Sec62-overproducing tumors.

A simple and reliable stir bar sorptive extraction-liquid chromatography procedure for the determination of chlorpromazine and trifluoperazine in human serum using experimental design methodology.

A sensitive and reproducible stir bar sorptive extraction and HPLC-UV detection method was used for the therapeutic drug monitoring of chlorpromazine and trifluoperazine in human serum. The separation was achieved using a C(18) column. The mobile phase consisted of methanol/sodium acetate buffer (pH 4.1; 0.1 M) (95:5, v/v) including 0.5% triethylamine. This miniaturized method can result in faster analysis, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. In the second part, stir bar sorptive extraction/HPLC-UV method was optimized by a chemometrics approach. An experimental design was therefore used to evaluate the statistically influential and/or interacting factors, among those described in the literature, and to find the best extraction and desorption conditions. Optimal sample volume of 1 mL, extraction time of 24 min at 31°C with pH 8.1 were obtained in a screening 2(5) half fractional factorial design followed by a Box-Behnken design. For the desorption conditions, a Box-Behnken design showed that the best conditions were 150 µL mobile phase for 20 min at 50°C. The optimized method was repeatable (CV<10%, linear (LOQ-500 ng/mL)), with the LOQs equal to 0.7 and 1.5 ng/mL for chlorpromazine and trifluoperazine, respectively.

Niveles sanguíneos de los neurolépticos / Serum levels of neuroleptics

La determinación de los niveles sanguíneos de la drogas antipsicóticas podría, eventualmente, proporcionar: una guía para determinar las dosis terapéuticas óptimas y un margen de seguridad dentro de un rango de dosis, una manera de discriminar a los pacientes refractarios, la información farmacocinética necesaria para ajustar adecuadamente las dosificaciones, la posibilidad de predecir los efectos terapéuticos y una ayuda en la selección de la droga para cada caso en particular. Evidentemente, estos puntos son de una gran trascendencia para la clínica, lo que ha dado lugar a un creciente interés en la investigación de las relaciones entre los niveles sanguíneos de los neurolépticos y diversas respuestas: la antipsicótica, la extrapiramidal y la endócrina. En el presente documento se revisan, en primer término, las técnicas de laboratorio que se han utilizado con mayor frecuencia para la cuantificación de las concentraciones de los neurolépticos en las muestras biológicas: cromatografía, radio-inmunoanálisis y ensayo de radio-receptor. En segundo lugar se analiza la información disponible acerca de los antipsicóticos de uso más frecuente en nuestro medio, abordándose aspectos farmacocinéticos, estudios referentes a las relaciones entre los niveles sanguíneos de la droga y los tres tipos de respuestas mencionadas, así como la influencia de otras drogas en las concentraciones sanguíneas de los antipsicóticos. Finalmente, se comenta la información presentada y se señala su relevancia para la práctica clínica

Development and application of a specific radioimmunoassay for trifluoperazine N4'-oxide to a kinetic study in humans.

A hapten derivative of prochlorperazine N4'-oxide, 7-(3-carboxy-propionyl)-prochlorperazine N4'-oxide, was synthesized and coupled to bovine serum albumin. Immunization of New Zealand White rabbits with this conjugate generated antibodies which were utilized to develop a radioimmunoassay (RIA) for the quantitation of trifluoperazine N4'-oxide (TFPNO). This assay enabled for the first time the quantitation of TFPNO in plasma, and 20 pg can be measured in a 200-microliter plasma sample with a coefficient of variation less than 5%. Statistically, indistinguishable results were obtained by the assay procedure with or without the selective extraction of TFPNO, and also in the presence or absence of an excess of trifluoperazine (TFP) and other major metabolites, such as trifluoperazine sulfoxide and 7-hydroxytrifluoperazine. This RIA and a previously reported RIA for TFP were used to directly determine plasma concentrations of TFP and TFPNO after administration of a single 5-mg p.o. dose of TFP to six healthy male volunteers. The mean +/- S.D. for the peak concentration, time to peak concentration, area under the curve from 0 to 72 hr and the elimination T1/2 for the terminal portion of the plasma concentration vs. time curve for TFPNO were found to be 3.7 +/- 0.7 ng/ml, 3.2 +/- 0.4 hr, 51.1 +/- 9.8 ng X hr/ml and 26.9 +/- 14.0 hr, respectively, whereas the corresponding values for TFP were found to be 2.7 +/- 0.7, 3.1 +/- 0.8, 42.2 +/- 6.9 and 21.9 +/- 6.5, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The strategy and value of neuroleptic drug monitoring.

The concept of monitoring neuroleptic drugs by means of chemical or pharmacological assays is considered from a historical and strategic point of view. The objectives of compliance control, reduction of long-term exposure, titration to a therapeutic range, and solution of medicolegal problems are separately considered. The relative merits of chemically specific (mainly chromatographic) and clinically specific (radioreceptor) assays are considered. An algorithm for interpretation of a neuroleptic concentration measurement is presented.

Radioimmunoassay for the sulfoxide metabolite of trifluoperazine and its application to a kinetic study in humans.

Antibodies were produced in rabbits immunized with 10-[[3-[4-(2-carboxyethyl)-1-piperazinyl]-propyl]]-2 -trifluoromethyl-10H-phenothiazine sulfoxide-bovine serum albumin conjugate. The subsequently developed radioimmunoassay (RIA) procedure enables, for the first time, the quantitation of the sulfoxide metabolite of trifluoperazine in the plasma of humans after administration of therapeutic doses of trifluoperazine, in which 60 pg of the sulfoxide metabolite in 200 microL of plasma can be measured with a CV of less than 3%. Similar results were obtained by this assay with or without a benzene extraction step and also in the presence or absence of a large excess of trifluoperazine and suspected major metabolites of trifluoperazine. This RIA procedure, together with a previously developed RIA for trifluoperazine was used to directly determine plasma concentrations of trifluoperazine and its sulfoxide metabolite after administration of a single, low, oral dose of trifluoperazine to five healthy volunteers. The rapidly appearing, relatively high concentrations of the sulfoxide metabolite are indicative of presystemic sulfoxidation. The mean plasma elimination half-life for the sulfoxide metabolic of trifluoperazine was 5.8 +/- 1.3 h.

Relative bioavailability of a commercial trifluoperazine tablet formulation using a radioimmunoassay technique.

The relative bioavailability of a new conventional tablet formulation (5 mg) of trifluoperazine dihydrochloride was studied in 24 healthy volunteers. Using a sensitive radioimmunoassay technique, plasma trifluoperazine concentrations were measured up until 24 h following ingestion of single 5-mg doses of trifluoperazine. The mean +/- SD for the peak concentration (Cmax), time to Cmax, area under the curve from 0 to 24 h (AUC240), and terminal elimination half-life following the administration of the test formulation were 2.15 +/- 1.07 ng/mL, 4.10 +/- 1.38 h, 21.04 +/- 11.92 ng X h/mL, and 9.5 +/- 7 h, respectively. Following the ingestion of the original trifluoperazine tablet formulation (5 mg) these same parameters were estimated to be 1.92 +/- 0.88 ng/mL, 4.02 +/- 1.10 h, 18.03 +/- 10.11 ng X h/mL, and 9.3 +/- 7 h, respectively. Large intersubject variations in Cmax and AUC240 were observed. The relative bioavailability of the test formulation was calculated to be 106.5 +/- 25.5%.

A bioequivalency study of two trifluoperazine tablet formulations using RIA and GC-MS.

Two sensitive analytical procedures, a radioimmunoassay (RIA) and a mass fragmentographic (GC-MS) method, were used to quantitate plasma trifluoperazine concentrations over 24 h in five healthy male volunteers following single 5 mg doses of two trifluoperazine tablet formulations (A and B) in a two-way cross-over design. Bioavailability in terms of area under the plasma concentration versus time curve to 24h or extrapolated to infinity, maximum plasma concentration and time to maximum plasma concentration using either RIA or GC-MS was not statistically significantly different from one formulation to the other. Also, there were no statistically significant differences between GC-MS and RIA values for AUC24(0) and Cmax for each of the two formulations examined. However, the mean AUC24(0) RIA/GC-MS ratios for formulations A and B were 3.1 and 3.4, respectively, while the mean Cmax RIA/GC-MS ratios were 1.7 and 2.1, respectively. These differences in AUC and Cmax are probably mainly due to the relative non-specificity of the RIA antiserum. Thus, where GC-MS is preferred for pharmacokinetic studies, both analytical procedures can be used for comparative single-dose bioequivalence studies of trifluoperazine. However, both the methods should be tested in patients in order to establish the suitability of one procedure over the other for the study of plasma level versus clinical response correlations.

Binding of phenothiazine neuroleptics to plasma proteins.

The binding of chlorpromazine, trifluoperazine, perphenazine, desipramine, propranolol and salicylic acid to human plasma and isolated plasma proteins was studied using equilibrium dialysis. Unlike salicylic acid, an acidic compound only bound to human serum albumin, the basic drugs were bound to all plasma protein fractions studied (albumin, alpha 1-acid glycoprotein, lipoproteins, gamma-globulins) with alpha 1-acid glycoprotein an important binding protein for each of them. The interaction of chloropromazine, perphenazine and trifluoperazine with alpha 1-acid glycoprotein was studied using Scatchard analysis. The primary class of binding sites revealed a low capacity (n = 0.5-1) and a high affinity (K = 10(5)-10(6) M-1) for the phenothiazines. The interaction of chlorpromazine, perphenazine and trifluoperazine with albumin was of the high capacity-low affinity type. In binding studies using plasma obtained from healthy volunteers, alpha 1-acid glycoprotein was found to be a very important binding protein for the basic drug studied with the exception of desipramine. This shows that results derived from binding studies using isolated protein fractions should be interpreted with caution.

Kinetics of oral trifluoperazine disposition in man.

The disposition of trifluoperazine (TFP) was studied in five healthy volunteers following oral administration of a 5 mg tablet. Using a very sensitive GC-MS technique plasma TFP concentrations were measured up until 24 h following drug ingestion. Peak plasma concentrations varied widely (range 0.53-3.09 ng ml-1) and were reached 2.8 +/- 0.5 h following ingestion of the TFP tablet. The apparent terminal elimination half-life of TFP was 12.5 +/- 1.4 h. The area under the plasma concentration-time curve differed widely between subjects (range: 5.9-17.6 ng ml-1 h) suggesting large individual differences in the extent of presystemic TFP elimination.

Radioimmunoassays for phenothiazine drugs and their major metabolites in plasma.

Radioimmunoassay procedures have been developed for some phenothiazine drugs and their major metabolites which are capable of quantitating 0.3 ng/ml or less in a 200 microliter plasma sample with a coefficient of variation less than 3%. They were developed with a view to their use in pilot investigations of plasma level versus clinical response correlations. The development of the procedures for trifluoperazine are described in this workshop. Antisera to trifluoperazine have been raised in New Zealand white rabbits to several different types of immunogens, where there was variation in the length and nature of the N-10 side chain attached to the phenothiazine nucleus, as well as in the number of hapten residues coupled to bovine serum albumin. An improved radioimmunoassay for trifluoperazine has been developed which showed cross-reactivities to the N-desmethyl and 7-hydroxy metabolites of trifluoperazine of the order of 12 and 11%, respectively, in comparison to the 26 and 24% cross-reactivity, respectively, for the same metabolites by a previously reported procedure.

Low serum neuroleptic levels predict relapse in schizophrenic patients.

Relapse occurs in a substantial proportion of schizophrenic patients treated with neuroleptics. The determinants of relapse have been elusive. In our study, low serum neuroleptic levels identified patients who had a relapse during a six-month period. Neuroleptic levels were measured by radioreceptor assay in 61 schizophrenic men and their clinical status was assessed in the subsequent six months. Ten patients had relapses, four showing a worsening of chronic psychotic symptoms and six showing eruption of psychotic symptoms after a period of remission. These ten patients had significantly lower normalized neuroleptic levels than those whose conditions remained stable. The lowest neuroleptic levels occurred in patients who had relapses after a period of remission. Serum neuroleptic levels in drug-responsive patients appear to be a critical determinant of remission. If these observations are replicated, a rational basis may be provided for prescribing and monitoring neuroleptic treatment and perhaps for preventing relapse.

A GLC-nitrogen phosphorous detector assay for trifluoperazine in plasma.

A GLC-nitrogen phosphorous detector (NPD) method for the quantitative determination of trifluoperazine in plasma is described. It depends on an organic extraction of trifluoperazine and the internal standard prochlorperazine from basified plasma. Following the extraction, the organic solvent is evaporated to dryness and the residue is reconstituted in a small volume of methyl alcohol. GLC analysis of aliquots of the methanolic solution using a NPD permitted the determination of 0.5 ng/ml of trifluoperazine in plasma. Standard curves for trifluoperazine over a concentration range of 0.5 to 15 ng/ml of plasma were linear with an overall variation coefficient of 5.3%. In isolated samples obtained from a normal healthy volunteer, application of this method to plasma concentration determinations after oral administration of a 5-mg trifluoperazine tablet, is demonstrated and compared with the concentrations obtained by GLC-mass spectrometry and radioimmunoassay procedures.

A gas chromatographic mass spectrometric assay for plasma trifluoperazine concentrations following single doses.

A gas chromatography mass spectrometric assay using selected ion monitoring is described which permits the determination of trifluoperazine in plasma at concentrations ranging from 0.078 to 5.0 ng ml-1. It relies on the extraction of trifluoperazine and the internal standard, prochlorperazine or the tetradeuterated analogue of trifluoperazine, from basified plasma with a n-pentane/2-propanol solvent mixture. Following the evaporation of organic solvent, the residue is reconstituted in a small volume of methanol. Suitable aliquots were analysed by the combined technique of gas chromatography/electron impact mass spectrometry with a data system. The described procedure is specific and enables the quantitation of 78 pg ml-1 of the drug with a coefficient of variation less than 7%. The method has demonstrated sufficient sensitivity to permit pharmacokinetic and bioavailability studies after a single 5 mg oral dose.

GLC analysis of trifluoperazine in human plasma.

This report describes a sensitive gas chromatographic procedure for the measurement of trifluoperazine in human plasma. Trifluoperazine was extracted into heptane-2-propanol by a two-step procedure and analyzed directly without derivatization. Prochlorperazine was employed as an internal standard because its structural and extraction characteristics were similar to trifluoperazine. The use of a nitrogen detection system reduced the number of interfering peaks. The within-day coefficient of variation in the methods, over a 0.2-20-ng/ml concentration range was 9.4%.

Radioimmunoassay for trifluoperazine in human plasma.

1 A new sensitive and rapid radioimmunoassay procedure for the determination of the plasma concentrations of the neuroleptic drug trifluoperazine is described. 2 The antiserum developed for trifluoperazine cross-reacted with N-desmethyltrifluoperazine and 7-hydroxytrifluoperazine to the extent of 26 and 24% respectively but its cross-reactivity with commonly co-administered tricyclic antidepressants and antianxiety agents tested was negligible. 3 The assay, based on the above antiserum, enabled the quantitation of 50 pg of the drug in 200 microliters of plasma with a coefficient of variation of about 2% and therefore should be applicable for singly dose pharmacokinetic and bioavailability studies. It should be applicable to therapeutic monitoring of the drug in patients.