ß-Phenylethylamine and various monomethylated and para-halogenated analogs. Acute toxicity studies in mice.

Phenylethylamine's acute toxic effects in a population of adult (10 to 12 weeks old; ∼30 g) Swiss male albino mice are significantly increased by para-position aromatic ring halogenation. LDLO, LD50, and LD100 values (mg/kg; x ± SEM) for p-F- (116.7 ± 3.3, 136.7 ± 1.7, and 160.0 ± 2.9), p-Br- (126.7 ± 3.3, 145.0 ± 2.9, and 163.3 ± 3.3), p-Cl- (133.3 ± 3.3, 146.7 ± 1.7, and 165.0 ± 2.9), and p-I-PEA (133.3 ± 3.3, 153.3 ± 1.7, and 168.3 ± 1.7), compared to PEA 203.3 ± 3.3, 226.7 ± 4.4, and 258.3 ± 8.8). Like PEA, the difference between LDLO and LD50, and LD50 and LD100 for individual amines were similar and in the range (10 to 20%). Toxicity variation between the various p-halogenatedPEAs also fell within a relatively narrow range (as a group: LDLO 116.7 ± 3.3 to 133.3 ± 3.3, LD50 136.7 ± 1.7 to 153.3 ± 1.7, and LD100 160.0 ± 2.9 to 168.3 ± 1.7 mg/kg). PEA methylation, (exception of its α-methyl derivative), results in relatively modest changes in acute toxicity. LDLO, LD50, and LD100 values (mg/kg; x ± SEM) for N-Me- (176.6 ± 3.3, 200.0 ± 2.9, and 221.7 ± 3.3), p-Me- (183.3 ± 3.3, 206.7 ± 3.3, and 225.0 ± 2.9), o-Me- (210.0 ± 5.8, 233.3 ± 3.3, and 258.3 ± 1.7), and ß-MePEA (220.0 ± 5.8, 243.3 ± 4.4, and 278.3 ± 44). Similar to PEA, and the p-HPEAs, the difference between LDLO and LD50 and LD50 and LD100 values for individual amines fell within a relatively narrow range (10 to 20%). Variation in toxicity among the methylatedPEAs also fell within a limited range (as a group: LDLO 176 ± 3.3 to 220 ± 5.8, LD50 200.0 ± 2.9 to 243.3 ± 4.4 and LD100 221.7 ± 3.3 to 278.3 ± 4.4 mg/kg). With the exception of PEA's methyl derivative (amphetamine) all the amines studied are rapidly metabolized by monoamine oxidase. This pharmacokinetics difference would help to explain the markedly higher amphetamine toxicity [(LDLO, LD50 and LD100 (mg/kg; x ± SEM) of 21.3 ± 0.9, 25.0 ± 0.6, and 29.3 ± 0.7, respectively)].

Behavioral effects of ß-phenylethylamine and various monomethylated and monohalogenated analogs in mice are mediated by catecholaminergic mechanisms.

The effects of the administration [intraperitoneally, 15 and 75 mg/kg, except α-MePEA (amphetamine, AMPH) at 5 and 10 mg/kg] of ß-phenylethylamine (PEA), its methylated (o-Me-, p-Me-, α-Me-, ß-Me-, N-Me-, p-OMe-, N,N-di-Me-, and 3,4-diOH-N-Me-), para-halogenated (Br-, Cl-, F-, and I-), and other derivatives for example, p-OHPEA (p-tyramine), on Swiss male albino mice caged behavior fall into 3 broad categories. (1) N,N-diMe-, 3,4-diOH-N-Me-, and o-MePEA tend to reduce the behavioral activity, (2) p-OH and p-IPEA were without noticeable effects, and (3) the remaining compounds increased locomotor activity, produced hyperexcitability and fighting, jumping and vocalization, and convulsion in a graded manner (listed in increasing order p-OMe-, ß-Me-, p-Cl-, p-Br-, p-F-, p-Me-, and N-MePEA, PEA itself and α-MePEA). The latter compound (amphetamine) being the most potent among them; equieffective but with lower potency were p-MePEA, N-MePEA, and PEA itself. The effects of PEAs upon group cage behavior were increased by pretreatment with pargyline (1.5 hours; 15 mg/kg) and decreased after reserpine or haloperidol [4 hours and/or 24 hours (2.5 and/or 2.5 mg/kg) and 1 hour (1 mg/kg), respectively], reaching full suppression with the double-dose regimen of reserpine and single dose of haloperidol. As expected, none of these substances by themselves were noticeable changed group mice activity or stereotypic behavior. The effects of test amines and catecholamine-modulating agents on stereotypy were assessed by rating the sequentially occurring behaviors: increased exploratory behavior with increased sniffing; occasional side-to-side head weaving; paw-licking and other grooming; gnawing, fighting and continuous side-to-side head weaving, and periodic episodes of "popcorn" behavior, during which all mice in the cage ran, jumped, and vocalized. In general, rank efficacy in eliciting stereotype aligned with rank efficacy in affecting group cage behavior. Our results show that a number of as yet little studied monomethylated and monohalogenated PEA analogs share a similar behavioral profile with PEA and AMPH. Behavioral changes observed appear to be, at least in part, mediated by catecholaminergic mechanism as they are modulated by drugs known to influence catecholamine activity. PEA analogs provide a large number of clinically useful drugs; whether further studies on these novel amines will lead to the rational design of newer, safer, and effective PEA-class drugs remains to be seen.

Analgesic effects of ß-phenylethylamine and various methylated derivatives in mice.

Administration of ß-phenylethylamine (PEA), the simplest endogenous neuroamine, and various methylated PEA derivatives including α-methyl PEA (amphetamine, AMP) elicits analgesia in mice. Five or 20 min after intraperitoneal PEA injection of as little as 6 mg/kg resulted in an increased latency response time (from 2.4 ± 0.4 to 8.5 ± 2.3 or 7.0 ± 3.0 s, respectively) to the thermal stimulus (hot-plate test), which reached statistical significance at the 15 mg/kg (20 min; 13.1 ± 0.4 s) or 25 mg/kg dose (5 min; 15.3 ± 4.1 s). This PEA effect, was dose-dependent (albeit non-linear: 6, 12, 15, 25, 50 and 100 mg/kg), reached the cut-off time of 45 s at the upper PEA dose (5 min), and it was consistently enhanced by pretreatment with the monoamine oxidase inhibitor pargyline (P). Methylated PEA derivatives (15 and 100 mg/kg dose) produced various degrees of analgesia (in decreasing order p-Me PEA > PEA > N,N-diMe PEA > N-Me PEA) which, likewise to PEA itself, were consistently increased by P and declined over time (mice tested 5, 20 and 60 min after amine injection); small but statistically significant o- and ß-Me PEA antinociceptive effects (5 min) were observed only at the higher dose (in the presence of P for ß-Me PEA). A small analgesic effect was observed after the administration of AMP (5 or 10 mg/kg) which failed, even after P, to reach statistically significance. Independent of the amine and concentration tested, individual compound's antinociceptive properties were reliably increased by P (exception of AMP), decreased by reserpine (R) or haloperidol (H), and remained essentially unchanged after naloxone (N) administration suggesting the involvement of catecholamines, but not opioid peptides, in their observed analgesic effects. Injection of P + N produced results similar to those seen after P alone. Under the experimental conditions described neither P, R, H or N had any effects by themselves. These findings suggest additional understanding of the mechanism of action responsible for the analgesic effects of these amines would be of interest, leading further to controlled studies on their alleged usefulness as weight reducing agents and sport performance enhancers.

Rat brain-uptake index for phenylethylamine and various monomethylated derivatives.

Phenylethylamine and its monomethylated derivatives p-methylphenylethylamine, α-methylphenylethylamine, phenylethylamine itself, N-methylphenylethylamine, o-methylphenylethylamine, and ß-methylphenylethylamine, readily cross the blood-brain barrier showing a brain-uptake index (%) ± SD (water considered 100 %), of 108 ± 11, 98 ± 14, 83 ± 6, 78 ± 11, 62 ± 7 and 56 ± 6, respectively (injection of tritiated water and 100 µg standard amine, which was measured by gas-liquid chromatography). Similar brain-uptake index values (determined by double isotope counting) were obtained for phenylethylamine and α-methylphenylethylamine (amphetamine) after the injection of tritiated water and C(14)-labeled amine (either 3 µg or when added 100 µg standard compound), suggesting that they entered the brain via passive diffusion. Accordingly, both amines distributed rather evenly in the various rat brain areas examined: uptake index (%) ± SD (double isotope counting; non-, and diluted labeled amine) for phenylethylamine (89 ± 8 and 78 ± 7, 83 ± 9 and 86 ± 9, 96 ± 6 and 84 ± 7) and for α-methylphenylethylamine (88 ± 11 and 87 ± 9, 93 ± 14 and 87 ± 11, 97 ± 12 and 87 ± 9) for the cerebellum, frontal cortex, and striatum, respectively. These results will aid a greater understanding of the pharmacological and behavioral effects observed after the administration of phenylethylamine and methylphenylethylamine derivatives.

A review of the etiology, associated comorbidities, and treatment of orthostatic hypotension.

The magnitude of increase in systolic blood pressure in response to the shift from supine to upright posture is considered to reflect the adequacy of orthostatic regulation. Orthostatic integrity is largely maintained by the interaction between the skeletal muscle pump, neurovascular compensation, neurohumoral effects, and cerebral blood flow regulation. Various physiological states and disease conditions may disrupt these mechanisms as seen in vasovagal syncope, dysautonomic orthostatic intolerance, and postural orthostatic tachycardia syndrome. Orthostatic hypotension (OH) and decreased cerebral blood flow are strongly related. Even subclinical OH has been associated to different degrees with impaired cognitive function, decreased effort, reduced motivation, increased hopelessness, and signs of attention-deficit hyperactivity disorder and dementia, diabetes mellitus, and Parkinson disease. Furthermore, subclinical levels of inadequate blood pressure regulation in response to orthostasis have been linked to increased depression and anxiety and intergenerational behavioral sequelae between mother and child. Identifying causes of subclinical and clinical OH is critical in improving quality of life for both children and older adults. A better understanding of the underlying causes responsible for the etiology of OH could lead to a rational design of novel effective therapeutic regimens for the treatment of this condition and associated comorbidities.

Changes in plasma methionine-enkephalin levels associated with a cluster headache episode.

Eighteen male cluster headache (CH) inpatients within a CH series participated in this research. Blood samples were drawn from patients at least 6-hour pain-free after the last acute CH episode and then shortly prior (SP), during, and soon after (SA) a new acute CH attack. Three healthy male, age-comparable drug-free volunteers served as controls; 5 samples were obtained from each of these individual over a 24-hour period. Individual patient's methionine-enkephalin (MET) plasma concentration showed significant changes, and in some subjects, dramatic changes, during the different phases of a single CH episode. Peptide levels followed a general pattern of higher plasma concentration SP to an acute CH attack, followed by decreased levels during the attack itself, and falling even further SA the acute episode. Consistently, 16 of the 18 patients tested showed pre-CH peptide levels significantly higher (arbitrarily the authors considered values 20% or more as "significant") than their own values obtained during the acute CH pain phase, with observed differences reaching 80% or more in 7 of these individuals. For about half of these patients, peptide concentration during the acute CH episode was significantly above the control's range (68.2-87.6 pg MET/mL; control's circulating MET concentration remaining essentially unchanged during a 24-hour period). MET levels were further decreased in essentially all of the post-CH samples, with values falling within (n = 6) or even further below than those in the control's range (n = 11). Neither age, time of CH occurrence, nor patient's use of a number of medications known for failing to influence plasma MET degradation kinetics seemed to significantly influence MET levels. These results might help in the biochemical characterization of the actual phases of a CH episode. Developing drugs modulating MET bioavailability could lead to novel antinociceptive agents useful for the treatment of CH's associated pain.

Decreased plasma methionine-enkephalin levels in cluster headache patients.

Results from a longitudinal study (blood drawn at days 29, 64, 89,124, 142, and 182 of the protocol) shows that the concentration of platelet-poor plasma (PPP) methionine(5)-enkephalin (MET) in healthy, drug-free, white male individuals (n = 5) remains within a relatively narrow range, well within the experimental error of the analytical procedures used. Interindividual differences fail to reach statistical significance [x ± SD and range (MET picograms per mL of PPP) of 91.2 ± 15.1, 67.1-113.5; 69.6 ± 7.5, 66.1-90.1; 76.6 ± 12.6, 58.5-93.1; 86.8 ± 10.9, 76.3-107.4; and 84.5 ± 11.4, 68.9-103.4; for subjects 1-5, respectively]. MET levels were similar to those recorded from single samples obtained from a group of 24 white male, age-comparable, drug-free healthy volunteers [x ± SD and range (picograms of MET per mL of PPP) of 83.3 ± 15.1 and 57.4-119.1]. The controls' range for all the subjects (n = 29) was 57.4-119.1 pgMET/mL PPP. Compared with the controls, individual patients with cluster headache (CH) show a much wider variation in PPP MET levels (blood drawn at different time intervals, at least 10 samples per patient, over a period of 221-298 days), with many (slightly over half) of single values below the controls range; no single MET level was above the controls range [x ± SD and range (picograms of MET per mL of PPP) of 56.4 ± 27.7, 6.1-100.5; 72.6 ± 20.5, 43.0-113.0; 46.0 ± 28.5, 10.0-92.6; 53.6 ± 27.5, 13.0-101.0; 52.0 ± 26.1, 17.5-83.6; 63.5 ± 22.3, 21.7-91.3 for individuals A-F, respectively]. Although interindividual differences within the patients' group were not statistically significant, their peptide levels were significantly lower than those of controls. Neither the presence of unspecified "headaches between clinic visits" and "daily headaches" (patients E and F, respectively), nor the use of a number of drugs known to lack inhibitory activity upon the aminopeptidase-MET degradation reaction, seemed to significantly influence MET concentration. The results could lead to a better understanding of the etiology of the pain associated with CH, with the relative changes in plasma peptide perhaps reflecting the patients' vulnerability to such a condition. Pharmacological modulation of MET function may prove useful in the treatment of CH-associated pain, whether the development of such drugs could find useful pharmacological applications remains to be explored.

A review of orthostatic blood pressure regulation and its association with mood and cognition.

AIMS: This paper will review literature that examines the psychological and neuropsychological correlates of orthostatic blood pressure regulation. RESULTS: The pattern of change in systolic blood pressure in response to the shift from supine to upright posture reflects the adequacy of orthostatic regulation. Orthostatic integrity involves the skeletal muscle pump, neurovascular compensation, neurohumoral effects and cerebral flow regulation. Various physiological states and disease conditions may disrupt these mechanisms. Clinical and subclinical orthostatic hypotension has been associated with impaired cognitive function, decreased effort, reduced motivation and increased hopelessness as well as dementia, diabetes mellitus, and Parkinson's disease. Furthermore, inadequate blood pressure regulation in response to orthostasis has been linked to increased depression and anxiety as well as to intergenerational behavioral sequalae. CONCLUSIONS: Identifying possible causes and consequences of subclinical and clinical OH are critical in improving quality of life for both children and older adults.

Degradation kinetics of methionine5-enkephalin by cerebrospinal fluid: in vitro studies.

Changes in the levels or biochemistry of cerebrospinal fluid (CSF) neuropeptides with opioid-like properties have been suggested to reflect alterations in specific biological processes. We have determined various kinetic parameters for methionine-enkephalin (MET) degradation by CSF samples from nonneurological patients. Study subjects included 9 males (51-67 years of age) and 5 females (47-61 years of age). Aliquots, removed from an incubation vessel containing buffer, CSF, and peptide [tyr-3',5'-H(N)MET], were analyzed for tyrosine and other degradation products. Essentially all of the labeled tyrosine from the added MET was recovered as free amino acid after 60 minutes of incubation (1:2 ratio, vol:vol; optimum pH 7.4; and temperature 37°C); other possible peptide metabolites (>3%) were not detected. Irrespective of age or gender, the peptide's degradation half-life and initial velocity values were in a limited range; t1/2 26.2 ± 5.5 and 20.8-33.8 minutes, and Iv 0.03 ±0.01 and 0.02-0.03 pg of peptide per milligram protein per minute. Km and Vmax values were 0.19 ± 0.02 and 0.17-0.21 mM, and 9.8 ± 2.2 and 7.6-12.0 µmol·L·min, respectively. Neither CSF sample storage time (up to a year) nor repeated freezing and thawing (up to 3 times over a year) altered the kinetics or products of this reaction. These preliminary findings might serve as reference values when conducting similar studies using CSF from patients diagnosed with specific neurological conditions; significant alterations in MET degradation profile in such a population could provide valuable biological markers for diagnostic and treatment purposes.

Etiology and risk factors for developing orthostatic hypotension.

Orthostatic hypotension (OH) is regarded as a decrease primarily in systolic blood pressure on changing position from supine to erect. Based on clinical criteria, it is characterized by a decrease in systolic pressure of 20 mmHg and diastolic pressure of 10 mmHg within 1 to 3 minutes of standing after being supine. It is most prevalent in, although not limited to, the elderly population and is characterized by a variety of problems, including diminished cognition and disturbed emotion along with gate problems, falls, and brain and cardiovascular difficulties. Although often seen as an age-related condition, occurrence of OH is also associated with a number of autonomic nervous system neurodegenerative disorders. Medications may play a direct role in the risk of triggering OH; these drugs include, but are not limited to, agents used in the treatment of hypertension, myocardial ischemia, psychosis and schizophrenia, depression, Alzheimer and Parkinson disease as well as a vaccine approved for the prevention of cervical cancer. Most of these agents increase the risk for triggering OH through varying vasodilative mechanisms or through sympathetic nervous system interruption; for other drugs, no mechanism of action has been identified. These factors should be considered when diagnosing OH and when prescribing remedies for both patients with OH and those without OH; medication's contributions to the severity and/or risk of developing OH could limit their use. However, their effects could be attenuated or even eliminated by modifying drug dosages.

Bacitracin-sensitive aminopeptidase(s) degradation of methionine(5)-enkephalin by human brain putamen and hippocampus preparations: inhibition by phenothiazine drugs.

Select phenothiazine drugs significantly decrease, in a dose-dependent manner, the rate of methione-enkephalin (MET) degradation by discrete human brain areas, for example, putamen and hippocampus. This pentapeptide is rapidly, and essentially completely, hydrolyzed at the tyrosine-glycine bond by bacitracin-sensitive aminopeptidase(s) (AP); neither dipeptidyl peptidase(s) (N-carboxyphenylmethyl leucine and captopril) nor peptidyl dipeptidase(s) (thiorphan) inhibitors altered the kinetics of MET degradation. Half-life (t1/2) and initial velocity (Iv) of this reaction were significantly increased and decreased, respectively, by fluphenazine > prochlorpherazine > chlorpromazine > thioridazine > promethazine > ethopropazine; brain A hippocampus (t1/2, control 2.8 and 60.2, 22.9, 15.4, 10.0, 9.9, and 4.8 minutes; and Iv, control 59.0 and 3.1, 10.7, 21.3, 22.8, 23.8, and 36.9 pg MET/mg brain tissue/minute) and putamen (t1/2, control 2.5 and 52.4, 19.9, 12.4, 8.2, 8.4, and 4.1 minutes; and Iv, control 53.1 and 2.7, 9.3, 17.1, 18.6, 20.1, and 31.7 pg MET/mg brain tissue/minute). Bacitracin was used for comparison purposes; hippocampus (t1/2 and Iv of 64.2 minutes and 4.3 pg MET/mg brain tissue/minute, respectively). Results using brain B tissue followed a comparable pattern, providing similar results. Hippocampus and putamen samples from both brains showed similar Km (mean 25.2, muM; range, 23.3-27.2 mum), Vmax (mean 108 nmol/mg protein/minute; range, 103-115 nmol/mg), and IC50 values (bacitracin, mean 14.4 muM; range, 13.8-14.7 mum) for MET AP degradation. Neither the phenothiazines methotrimeprazine or trifluoperazine nor other commonly used nonphenothiazine antipsychotic tested, for example, clozapine, haloperidol, loxapine, molindone, sulpiride and thiothixine, significantly altered the kinetics of this reaction. The presence of the phenothiazine molecule appears to be necessary for AP inhibition; however, our results failed to show s correlation among chemical structure, pharmacologic profile, and tested compound ability to inhibit MET degradation. This research provides initial information that could lead to the rational design of agents capable of modulate the bioavailability of enkephalins and other AP-metabolized biologically active compounds. Whether their development could find useful pharmacologic applications remains to be explored.

Degradation kinetics of methionine5-enkephalin by select brain areas from patients with chronic schizophrenia.

After 10-minute incubation of [3H]-tyrosine methionine-enkephalin (MET) with 100,000 x g supernatant from select brain regions of patients with chronic schizophrenia (n = 3), essentially all of the labeled tyrosine was recovered as the free amino acid. Initial velocity and half-life of MET degradation obtained from different brain areas (limbic system, thalamus, basal ganglia, cerebellum, and cortex) of individual brains or from equivalent sections from different brains were scattered and considerable spread out (brains A, B, and C: 21.7-60.2 and 2.1-14.3, 25.6-88.7 and 1.6-14.1, 24.5-56.1 and 2.6-14.3 pg MET/mg brain tissue/min and min, respectively; brains A-C range, 21.7-88.7 pg MET/mg brain tissue/min and 1.6-14.3 min, respectively). These results failed to identify consistent differences in peptide degradation kinetics between the various brains areas studied from the same individual or from equivalent section from different subjects. MET metabolic rate was pH and temperature-dependent (optimum 7.4 degrees C and 37 degrees C), reduced by the aminopeptidase inhibitors puromycin, bacitracin, and bestatin, and to a lesser extent by thioridazine. However, peptide metabolism was not significantly affected by differences in tissue storage time or repeated freezing and thawing; by preincubation with N-carboxymethyl phenyl leucine, captopril, or thiorphan (dipeptidyl peptidase[s] or peptidyl dipeptidase[s] inhibitors, respectively); or by the many different drugs used by the patients with chronic schizophrenia. Our findings, although of a preliminary nature and generally similar to those recently reported for comparable studies on nonneuropsychiatric patients, provide a much needed understanding of the mechanisms regulating brain MET metabolism. Whether these results may contribute to the rational design of pharmacologic strategies for the treatment of pathologies associated with alterations in the enkephalinergic system needs further research.

Biological correlates of migraine and cluster headaches: an overview of their potential use in diagnosis and treatment.

Current diagnostic criteria for headaches are based on the International Classification for Headache Disorders, second edition, which is largely built on data obtained from clinical examinations and patients' medical histories. Despite decades of vigorous basic and clinical research, we still lack reliable clinical laboratory diagnostic markers for headaches, which clearly obstructs the physician's ability to optimize and follow the patient's response to treatment protocols as well as holds back the discovery and implementation of new therapeutic modalities. In this paper, we review and discuss current efforts to identify and characterize biochemical and immunological changes in biological fluids and tissue that may be specifically associated with the etiology and/or pathophysiology of migraine and cluster headaches; we also discuss some of the recent genetic findings and ion channel modulation studies that may help to distinguish among various headache populations.

Phenothiazine molecule provides the basic chemical structure for various classes of pharmacotherapeutic agents.

The chemical structure of phenothiazine provides a most valuable molecular template for the development of agents able to interact with a wide variety of biological processes. Synthetic phenothiazines (with aliphatic, methylpiperazine, piperazine-ethanol, piperazine-ethyl, or piperidine side-chain) and/or phenothiazine-derived agents e.g., thioxanthenes, benzepines, imonostilbenes, tricyclic antidepressants, dimetothiazine, and cyproheptadine have been effective in the treatment of a number of medical conditions with widely different etiology. These include various currently clinically used drugs for their significant antihistamic, antipsychotic, anticholinergic (antiparkinson), antipruritic, and/or antiemetic properties. They are also employed, although to a minor extent, as antidepressants, antispasmodics, analgesics, and antiarrhythemics. Some of these agents are also useful as anti-inflammatory, coronary vasodilator, radioprotective, sedative, antitussive, and skeletal muscle-relaxing medication. Still, others show different degrees of effectiveness as antibacterials, anthelmintics, antimalarials, or local anesthetics; a few are valuable in the control of acute migraine attacks and intractable hiccough. Adding to the seemingly ever-expanding therapeutic use of phenothiazine derivatives, a number of "old" and newly synthesized compounds e.g., "half-mustard-type" and benzo[alpha]phenothiazines, appear to be helpful as multidrug resistance modifiers, a property of particular importance in cancer chemotherapy. Some phenothiazines inhibit human plasmatic leucine-enkephalin aminopeptidase(s), enzymes known to regulate the turnover rate of a wide range of bioactive substances. These findings could lead to the design of new therapeutic treatment modalities for conditions such as Alzeimer's and Creutzfeldt-Jakob disease. Hopefully, this work will help to the rational design of new and improved pharmacological approaches based on a better understanding of the correlation between chemical structure, pharmacodynamic properties, and pharmacological activity of various phenothiazines and phenothiazine-derived classes of drugs.

Inhibition of human plasma leucine5-enkephalin aminopeptidase hydrolysis by various endogenous peptides and a select number of clinically used drugs.

We identified a number of clinically used drugs and biologically active endogenous peptides able to significantly decrease the rate of human plasmatic aminopeptidase (AP) leucine-enkephalin (LEU) degradation. Bacitracin, bestatin, fluvoxamine, and each of 4 peptides tested significantly increased, in a dose-dependent manner (10-10 M), LEU degradation half-life (t1/2) in each of 5 plasma samples studied. Each sample was obtained by pooling equal volume of 6 randomly selected, individual plasmas (4 male and 2 female healthy, drug-free volunteers). Thirty subjects (20 females and 10 males) participated in this study. With the exception of fluvoxamine, this inhibitory effect was lacking in various other commonly used drugs with widely different chemical structures and pharmacological profiles, eg, antidepressants (SSRIs, imipramine-like tricyclics, MAOIs), acute antimigraine agents (triptan class drugs), the nonselective beta-adrenergic antagonist propranolol, and serotonin receptor agonists and antagonists. Agents (concentration 10 M used as illustration), listed in decreasing order of LEU-AP inhibitory activity: substance P > angiotensin III > methionine-enkephalin > angiotensin II > fluvoxamine > bestatin gave t1/2 values (+/- SD) of 39.3 +/- 1.1, 29.4 +/- 0.8, 28.3 +/- 0.8, 27.4 +/- 0.7, 24.5 +/- 1.5, and 23.6 +/- 0.9 minutes, respectively. Control, bacitracin, and fluphenazine (known LEU-AP inhibitors were used for comparison) values of 11.8 +/- 1.0, 31.3 +/- 0.7, and 19.6 +/- 1.0 minutes, respectively. As expected, these drugs significantly decreased the initial velocity of peptide degradation; Iv values (+/- SD) of: 0.17 +/- 0.1 (0.02 +/- 0.01), 0.23 +/- 0.2 (0.02 +/- 0.01), 0.25 +/- 0.2 (0.02 +/- 0.01), 0.26 +/- 0.2 (0.03 +/- 0.01), 0.31 +/- 0.1 (0.03 +/- 0.01), and 0.33 +/- 0.1 (0.03 +/- 0.01), respectively; control, bacitracin, and fluphenazine: 1.10 +/- 0.3 (0.12 +/- 0.03), 0.20 +/- 0.1 (0.02 +/- 0.01), and 0.82 +/- 0.2 (0.08 +/- 0.02) pg LEU/min (pg LEU/mg protein/min), respectively. Results emphasize the selective nature of chemical structures required to significantly inhibit AP activity and provided information that could help the rational design of agents with high specificity in a biologic milieu containing multiple peptidases. In this case, targeted modulation of the bioavailability of LEU and other endogenous AP-degraded hormonal and nonhormonal peptides could be useful in the treatment of the pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.

Human natural killer cell lysis of Salmonella typhi intracellularly-infected U937 cells.

Peripheral blood mononuclear cell (PBMC) cytotoxicity against S. typhi (wild type or mutant strain TYT1231)-infected U937 cells was significantly higher than its lytic effect against noninfected cells (control) at the various effector-to-target cell ratio used (30:1, 50:1 and 70:1). Natural killer cell activity [expressed as % specific lysis (mean +/- SEM); 30:1 (25.4 +/- 3.6, 25.1 +/- 4.2 and 16.3 +/- 3.3); 50:1 (27.8 +/- 3.7, 26.7 +/- 4.5 and 20.9 +/- 2.9) and 70:1 ratio (33.2 +/- 5.9, 29.4 +/- 4.2 and 22.8 +/- 2.8), respectively] appeared to be dependent on such ratios and independent of the S strain studied. Most (80%) of individual samples tested showed at least a 20% specific lysis increase over their own control; essentially no changes or smaller increases in NKC activity were observed in all other samples. Similar results were obtained when using highly purified NKC (HPNKC) preparations as effector cells [NKC activity (mean +/- SEM); 5:1 (46.2 +/- 4.7, 43.2 +/- 5.0 and 25.2 +/- 2.3) and 10:1 effector-to-target cell ratio (49.3 +/- 4.9, 44.7 +/- 5.2 and 27.2 +/- 2.6, respectively)]. All individual samples tested showed at least a 20% specific lysis increase over their own control. These results show that S. typhi-infected U937 cells are a significantly better target for NKCs than control cells and indicate that intracellular bacteria survival capacity is not a critical factor for infected cells becoming a NKC target.

Lysis of salmonella typhi intracellularly infected U937 cells by human natural killer cells: effect of protein kinase inhibitors.

We examined the effect of Salmonella typhi (wild-type Ty2 and mutant strain TYT1231)-infected U937 cells on natural killer cell (NKC) cytotoxicity of peripheral blood mononuclear cells (PBMCs) and highly purified NKC (HPNKCs; CD16(+)/CD56(+) > 95%; the rest corresponding to CD3(+) T cells). We also analyzed the possible role of various protein kinases involved in natural cytotoxicity on these processes. PBMC cytotoxicity against S typhi-infected U937 cells was significantly higher (paired Student t test; P < 0.05) than its lytic effect against noninfected cells (control) at the various effector-to-target cell ratios used (30:1 [24.4 +/- 9.7, 25.1 +/- 11.8, and 17.5 +/- 8.6]; 50:1 [26.6 +/- 9.7, 26.7 +/- 12.8, and 21.2 +/- 7.5] and 70:1 [32.4 +/- 14.4, 30.1 +/- 12.4, and 23.1 +/- 7.2], respectively). PBMC NKC activity seemed to be dependent on such ratios and was similar against both Salmonella strains studied. Approximately half of the individual samples tested (n = 12; 8 male and 4 female subjects of comparable age) showed at least a 20% specific lysis increase against their own control; essentially no changes or smaller increases in NKC activity were observed in all other samples. Similar results were obtained using HPNKCs as effector cells (5:1 ratio [38.9 +/- 12.3, 43.3 +/- 11.2, and 27.5 +/- 4.9] and 10:1 ratio [51.3 +/- 9.1, 46.1 +/- 9.8, and 37.7 +/- 15.5, respectively]). In general, specimens significantly lysed after incubation with PBMCs responded in a similar manner to a challenge with HPNKCs. PBMC and HPNKC cytotoxicity against S typhi wild-type-infected U937 cells was significantly decreased in a dose-dependent manner by the addition of genistein (50-200 micromol) or GFX (0.5-2.0 micromol) to the cytotoxicity assay mixture. NKC activity was almost completely inhibited at the highest genistein and GFX concentrations. In similar experiments, wortmannin (100-500 nmol) failed to inhibit PBMC cytotoxicity and significantly decreased HPNKC activity only at the highest concentration tested. These results show that in the process of NKC recognition and lysis of S typhi-infected U937 cells, there is not a requisite for full bacterial intracellular survival capacity and that S typhi-infected U937 cells are a significantly better target than noninfected U937 cells. NKC signaling pathways activated during the S typhi-infected U937 cell recognition and lysis process are mainly protein tyrosine kinase and protein kinase-C, and they can be blocked by the same protein kinase inhibitors known to inhibit natural cytotoxicity.

In vitro human plasma leucine(5)-enkephalin degradation is inhibited by a select number of drugs with the phenothiazine molecule in their chemical structure.

A number of drugs with the phenothiazine molecule in their chemical structure inhibit in a dose-dependent manner human plasmatic aminopeptidase leucine(5)-enkephalin (LEU) metabolism. Half-life peptide degradation was significantly increased by thioridazine > fluphenazine > As-1397 [10-(alpha-diethylaminopropionyl)phenothiazine] >/= promethazine >/= chlorpromazine (final drug conc. 10(-4) M); t1/2 (+/- SD) 21.2 +/- 1.1, 19.6 +/- 1.0, 17.2 +/- 0.9, 17.1 +/- 1.0, and 17.1 +/- 1.1 min, respectively. Control and bacitracin (known aminopeptidase inhibitor) values were 11.8 +/- 1.0 and 31.3 +/- 1.7 min, respectively. These drugs significantly decreased (listed in the same order) LEU degradation initial velocity; Iv (+/- SD) 0.77 +/- 0.2, 0.82 +/- 0.2, 0.92 +/- 0.3, 0.93 +/- 0.2, 0.94 +/- 0.3 pg LEU/min, respectively. Control and bacitracin 1.10 +/- 0.3 and 0.20 +/- 0.1 pg LEU/min, respectively. Values represent results from 5 samples, each obtained by pooling 6 individual plasmas (4 male and 2 female; n = 30 healthy, drug-free volunteers). However, neither the phenothiazines ethopropazine, methotrimeprazine, prochlorperazine and trifluoperazine nor the various commonly used heterocyclic antipsychotics tested, e.g., molindone, loxapine, clozapine, haloperidol, sulpiride and thiothixene inhibited plasma LEU degradation kinetics. Our results failed to show correlations between chemical structure, antipsychotic properties and ability to inhibit plasmatic aminopeptidase LEU degradation. Whereas, presence of the phenothiazine molecule appears to be necessary for enzyme inhibition, only five out of nine substituted phenothiazines tested exhibited this effect. Furthermore, there was a lack of correlation between phenothiazines antipsychotic properties and their capacity to inhibit aminopeptidase activity, a property shown by promethazine (antihistaminic) and As-1397 (selective butyrylcholinesterase inhibitor) but lacking in prochlorperazine and trifluoperazine. Our results provide information which could lead to the rational design of agents capable to modulate the bioavailability of enkephalin and other endogenous aminopeptidase-degraded peptides believed to be involved in the etiology and/or pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.