ß1- and ß1/ß2-adrenergic receptor antagonists block 6-nitrodopamine-induced contractions of the rat isolated epididymal vas deferens.

6-Nitrodopamine (6-ND) is an endogenous modulator of the contractility in the rat isolated epididymal vas deferens (RIEVD) and considered to be the main peripheral mediator of the emission process. Use of selective and unselective ß-adrenergic receptor antagonists has been associated with ejaculatory failure. Here, the effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline, and electric-field stimulation (EFS) were investigated. The selective ß1-adrenergic receptor antagonists atenolol (0.1 and 1 µï»¿M), betaxolol (1 µï»¿M), and metoprolol (1 µï»¿M) and the unselective ß1/ß2-adrenergic receptor antagonists propranolol (1 and 10 µï»¿M) and pindolol (10 µï»¿M) caused significant rightward shifts of the concentration-response curve to 6-ND (pA2 6.41, 6.91, 6.75, 6.47, and 5.74; for atenolol, betaxolol, metoprolol, propranolol, and pindolol), but had no effect on dopamine-, noradrenaline-, and adrenaline-induced contractions. The effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists at a higher concentration (atenolol 1 µï»¿M, betaxolol 1 µï»¿M, metoprolol 1 µï»¿M, propranolol 10 µï»¿M, and pindolol 10 µï»¿M) also reduced the EFS-induced RIEVD contractions in control, but not in RIEVD obtained from L-NAME-treated animals. The selective ß1-adrenoceptor agonist RO-363, the selective ß2-adrenoceptor agonist salbutamol, and the selective ß3-adrenoceptor agonist mirabegron, up to 300 µï»¿M, had no effect on the RIEVD tone. The results demonstrate that ß1- and ß1-/ß2-adrenoceptor receptor antagonists act as 6-ND receptor antagonists in RIEVD, further confirming the main role of 6-ND in the RIEVD contractility.

Pindolol potentiates the antidepressant effect of venlafaxine by inhibiting 5-HT1A receptor in DRN neurons of mice.

INTRODUCTION: Antidepressants increase the level of 5-HT in the somatodendritic region of the serotonergic dorsal raphe nucleus (DRN) neurons in the first few days of their usage, which, in turn, inhibits the serotonergic neurons locally. Pindolol may eliminate this inhibition when used in combination with antidepressants. MATERIAL AND METHODS: We aimed to determine the effect of pindolol on 5-HT1A receptor response in the DRN neurons, using voltage clamp recordings and prove the potentiation of antidepressant effect of venlafaxine by pindolol through behavior experiments. Balb/c mice, 28-35 days-old were used. RESULTS: 5-HT application (25 µM) induced an outward current by 23.36 ± 3.79 pA at the neurons in the dorsal subnucleus of DRN. This effect was inhibited by pre-administration of WAY-100135 (21 µM) and pindolol (10 µM) separately. The current induced by 5-HT and 8-OHDPAT have no statistically significance. 8-OHDPAT (30 µM) induced a 5-HT-like outward current, which was inhibited by pre-administration of pindolol (10 µM). Combination of venlafaxine (20 mg/kg/day) and pindolol (15 mg/kg/day) significantly reduced immobilization time when compared to the control group in tail suspension test and forced swim test without any significant change in locomotor activity. Administration of venlafaxine (20 mg/kg/day) alone or pindolol (15 mg/kg/day) alone did not significantly reduce immobilization time. CONCLUSION: Pindolol has the potential to prevent the inhibition of serotonergic neurons after antidepressant use. Hence, we, for the first time, demonstrated that pindolol can potentiate antidepressant effect of venlafaxine. In the mood disorders, pindolol is likely to increase the effectiveness of antidepressant drugs when given in combination.

MT-102 prevents tissue wasting and improves survival in a rat model of severe cancer cachexia.

BACKGROUND: Cachexia, a common manifestation of malignant cancer, is associated with wasting of skeletal muscle and fat tissue. In this study, we investigated the effects of a new first in class anabolic catabolic transforming agent on skeletal muscle in a rat model of cancer cachexia. METHODS: Young male Wistar Han rats were intraperitoneally inoculated with 108 Yoshida hepatoma AH-130 cells and once daily treated with 0.3 mg kg-1 , 3 mg kg-1 MT-102, or placebo by gavage. RESULTS: Three mg kg-1 d-1 MT-102 not only prevented progressive loss of fat mass (-6 ± 2 g vs -12 ± 1 g; P < 0.001); lean mass (+1 ± 10 g vs. -37 ± 2 g; P < 0.001) and body weight (+1 ± 13 g vs. -60 ± 2 g; P < 0.001) were remained. Quality of life was also improved as indicated by a higher food intake 12.9 ± 3.1 g and 4.3 ± 0.5 g, 3 mg kg-1 d-1 MT-102 vs. placebo, respectively, P < 0.001) and a higher spontaneous activity (52 369 ± 6521 counts/24 h and 29 509 ± 1775 counts/24 h, 3 mg·kg-1 d-1 MT-102 vs. placebo, respectively, P < 0.01) on Day 11. Most importantly, survival was improved (HR = 0.29; 95% CI: 0.16-0.51, P < 0.001). The molecular mechanisms behind these effects involve reduction of overall protein degradation and activation of protein synthesis, assessed by measurement of proteasome and caspase-6 activity or Western blot analysis, respectively. CONCLUSIONS: The present study shows that 3 mg kg-1 MT-102 reduces catabolism, while inducing anabolism in skeletal muscle leading to an improved survival.

Enhancing the efficacy of 5-HT uptake inhibitors in the treatment of attention deficit hyperactivity disorder.

Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common childhood behavioural disorders, the frontline treatments for which are drugs with abuse potential. As a consequence, there is an urgent need to develop non addictive drug treatments with equivalent efficacy. Preclinical evidence suggests that selective serotonin uptake inhibitors (SSRIs) are likely to be effective in ADHD, however clinical reports suggest that SSRIs are of limited therapeutic value for the treatment of ADHD. We propose that this disconnect can be explained by the pattern of drug administration in existing clinical trials (administration for short periods of time, or intermittently) leading to inadequate control of the autoregulatory processes which control 5-HT release, most notably at the level of inhibitory 5-HT1A somatodendritic autoreceptors. These autoreceptors reduce the firing rate of 5-HT neurons (limiting release) unless they are desensitised by a long term, frequent pattern of drug administration. As such, we argue that the participants in earlier trials were not administered SSRIs in a manner which realises any potential benefits of targeting 5-HT in the pharmacotherapy of ADHD. In light of this, we hypothesise that there may be under-researched potential to exploit 5-HT transmission therapeutically in ADHD, either through changing the administration regime, or by pharmacological means. Recent pharmacological research has successfully potentiated the effects of SSRIs in acute animal preparations by antagonising inhibitory 5-HT1A autoreceptors prior to the administration of the SSRI fluoxetine. We suggest that combination therapies linking SSRIs and 5-HT1A antagonists are a potential way forward in the development of efficacious non-addictive pharmacotherapies for ADHD.

The behaviour of some antihypertension drugs on human serum paraoxonase-1: an important protector enzyme against atherosclerosis.

OBJECTIVES: Paraoxonase-1 (PON1) enzyme is related to high-density lipoprotein (HDL), which is calcium dependent. It has essential roles such as protecting LDL against oxidation and detoxification of highly toxic substances. It is a significant risk to reduce the levels of this enzyme in patients with diabetes mellitus, cardiovascular diseases, hyperthyroidism and chronic renal failure. METHODS: Here, it was reported that the purification of human serum PON1 using straightforward methods and determination of the interactions between some antihypertension drugs and the enzyme. KEY FINDING: It was found that these drugs exhibit potential inhibitor properties for human serum PON1 with IC50 values in the range of 131.40-369.40 µm and Ki values in the range of 56.24 ± 6.75-286.74 ± 28.28 µm. These drugs showed different inhibition mechanisms. It was determined that midodrine and nadolol were exhibited competitive inhibition, but atenolol and pindolol were exhibited non-competitive inhibition. CONCLUSION: Usage of these drugs would be hazardous in some cases.

Measuring endogenous changes in serotonergic neurotransmission with [11C]Cimbi-36 positron emission tomography in humans.

Developing positron emission tomography (PET) radioligands for the detection of endogenous serotonin release will enable the investigation of serotonergic deficits in many neuropsychiatric disorders. The present study investigates how acute challenges that aim to increase or decrease cerebral serotonin levels affect binding of the serotonin 2A receptor (5-HT2AR) agonist radioligand [11C]Cimbi-36. In a randomized, double-blind, placebo-controlled, three-arm design, 23 healthy volunteers were PET scanned twice with [11C]Cimbi-36: at baseline and following double-blind assignment to one of three interventions (1) infusion of the selective serotonin reuptake inhibitor (SSRI) citalopram preceded by oral dosing of the 5-HT1AR antagonist pindolol, (n = 8) (2) acute tryptophan depletion (ATD) (n = 7) and (3) placebo (n = 8). Two-sample t-tests revealed no significant group differences in percent change of neocortical [11C]Cimbi-36 binding from baseline to intervention between placebo and citalopram/pindolol (p = 0.4) or between placebo and ATD (p = 0.5). Notably, there was a significantly larger within-group variation in 5-HT2AR binding after intervention with citalopram/pindolol, as compared with placebo (p = 0.007). These findings suggest that neither ATD nor a combination of citalopram and pindolol elicit acute unidirectional changes in serotonin levels sufficient to be detected with [11C]Cimbi-36 PET in neocortex. We suggest that the large interindividual variation in 5-HT2AR binding after citalopram/pindolol reflects that after an acute SSRI intervention, individuals respond substantially different in terms of their brain serotonin levels. Our observation has a potential impact for the understanding of patient responses to SSRI.

Monoaminergic system and depression.

Major depressive disorder is a severe, disabling disorder that affects around 4.7% of the population worldwide. Based on the monoaminergic hypothesis of depression, monoamine reuptake inhibitors have been developed as antidepressants and nowadays, they are used widely in clinical practice. However, these drugs have a limited efficacy and a slow onset of therapeutic action. Several strategies have been implemented to overcome these limitations, including switching to other drugs or introducing combined or augmentation therapies. In clinical practice, the most often used augmenting drugs are lithium, triiodothyronine, atypical antipsychotics, buspirone, and pindolol, although some others are in the pipeline. Moreover, multitarget antidepressants have been developed to improve efficacy. Despite the enormous effort exerted to improve these monoaminergic drugs, they still fail to produce a rapid and sustained antidepressant response in a substantial proportion of depressed patients. Recently, new compounds that target other neurotransmission system, such as the glutamatergic system, have become the focus of research into fast-acting antidepressant agents. These promising alternatives could represent a new pharmacological trend in the management of depression.

Modulation of serotonin and noradrenaline in the BLA by pindolol reduces long-term ethanol intake.

Repeated cycles of binge-like alcohol consumption and abstinence change the activity of several neurotransmitter systems. Some of these changes are consolidated following prolonged alcohol use and are thought to play an important role in the development of dependence. We have previously shown that systemic administration of the dual beta-adrenergic antagonist and 5-HT1A/1B partial agonist pindolol selectively reduces long-term but not short-term binge-like consumption of ethanol and alters excitatory postsynaptic currents in basolateral amygdala (BLA) principal neurons. The aim of this study was to investigate the effects of pindolol microinfusions in the BLA on long-term ethanol intake using the drinking-in-the-dark paradigm in mice. We also microinfused RU24969 (5-HT1A/1B receptor partial agonist) and CGP12177 (ß1/2 adrenergic antagonist) following long-term ethanol intake and determined the densities of 5-HT1A/1B receptors and ß1/2 adrenergic in the BLA following short-term (4 weeks) and long-term ethanol (12 weeks) consumption. We show that intra-BLA infusion of pindolol (1000 pmol/0.5 µl), RU24969 (0.3 and 3 pmol/0.5 µl) and CGP12177 (500 pmol/0.5 µl) produce robust decreases in long-term ethanol consumption. Additionally, we identified reduced ß1/2 adrenergic receptor expression and no change in 5-HT1A/1B receptor density in the BLA of long-term ethanol-consuming mice. Collectively, our data highlight the effects of pindolol on voluntary, binge-like ethanol consumption behavior following long-term intake.

ß2-adrenoreceptor Inverse Agonist Down-regulates Muscarine Cholinergic Subtype-3 Receptor and Its Downstream Signal Pathways in Airway Smooth Muscle Cells in vitro.

Mechanisms underlying ß2-adrenoreceptor (ß2AR) inverse agonist mediated bronchoprotectiveness remain unknown. We incubated ICI118,551, formoterol, budesonide, and formoterol plus budesonide, as well as ICI118,551 or pindolol plus formoterol, ICI118,551 plus forskolin, SQ22,536 or H89 plus formoterol in ASMCs to detect expressions of M3R, PLCß1 and IP3. The level of M3R in the presence of 10-5 mmol/L ICI118,551 were significantly decreased at 12 h, 24 h and 48 h (P < 0.05), and at 24 h were significantly reduced in ICI118,551 with concentration of 10-5 mmol/L, 10-6 mmol/L, 10-7 mmol/L, and 10-8 mmol/L (P < 0.05). The level of IP3 in 10-5 mmol/L ICI118,551 was significantly diminished at 24 h (P < 0.01), except for that at 1 h, neither was in the level of PLCß1. A concentration of 10-5 mmol/L ICI118,551 at 24 h showed a significant reduction of M3R level compared to formoterol (P < 0.01), budesonide (P < 0.01), and formoterol + budesonide (P < 0.05), but significant reduction of PLCß1 and IP3 was only found between 10-5 mmol/L ICI118,551 and formoterol at 24 h, but not in the comparison of budesonide or formoterol + budesonide. Pindolol and H89 could not inhibit the formoterol-induced expression of M3R (P > 0.05), but SQ22,536 significantly antagonized the formoterol-induced M3R expression (P < 0.05). In conclusions, ß2AR inverse agonist, ICI118,551, exerts similar bronchoprotective effects to corticosteroids via decreasing the expression of M3R and inhibiting the production of IP3.

The antihypertensive drug pindolol attenuates long-term but not short-term binge-like ethanol consumption in mice.

Alcohol dependence is a debilitating disorder with current therapies displaying limited efficacy and/or compliance. Consequently, there is a critical need for improved pharmacotherapeutic strategies to manage alcohol use disorders (AUDs). Previous studies have shown that the development of alcohol dependence involves repeated cycles of binge-like ethanol intake and abstinence. Therefore, we used a model of binge-ethanol consumption (drinking-in-the-dark) in mice to test the effects of compounds known to modify the activity of neurotransmitters implicated in alcohol addiction. From this, we have identified the FDA-approved antihypertensive drug pindolol, as a potential candidate for the management of AUDs. We show that the efficacy of pindolol to reduce ethanol consumption is enhanced following long-term (12 weeks) binge-ethanol intake, compared with short-term (4 weeks) intake. Furthermore, pindolol had no effect on locomotor activity or consumption of the natural reward sucrose. Because pindolol acts as a dual beta-adrenergic antagonist and 5-HT1A/1B partial agonist, we examined its effect on spontaneous synaptic activity in the basolateral amygdala (BLA), a brain region densely innervated by serotonin and norepinephrine-containing fibres. Pindolol increased spontaneous excitatory post-synaptic current frequency of BLA principal neurons from long-term ethanol-consuming mice but not naïve mice. Additionally, this effect was blocked by the 5-HT1A/1B receptor antagonist methiothepin, suggesting that altered serotonergic activity in the BLA may contribute to the efficacy of pindolol to reduce ethanol intake following long-term exposure. Although further mechanistic investigations are required, this study demonstrates the potential of pindolol as a new treatment option for AUDs that can be fast-tracked into human clinical studies.

NO-dependent attenuation of TPA-induced immunoinflammatory skin changes in Balb/c mice by pindolol, heptaminol or ATRA, but not by verapamil.

Recently a mouse skin carcinogenesis study reported that a ß-blocker carvedilol displayed antitumor-properties via antihyperplastic effects. However, the antihyperplastic mechanism is unclear as the ß-blocker is characterized with multiple pleiotropic effects including stimulation of endothelial NO release and verapamil-like calcium channel blocking activity. To investigate the nature and the origin of the antihyperplastic effects, we tested topical pretreatment with pindolol, heptaminol, ATRA or verapamil against Balb/c mouse ear skin hyperplasia that was induced by TPA. We found that pindolol, heptaminol or ATRA, but not verapamil, inhibited the TPA-induced immunoinflammatory skin changes in an NO-dependent manner, which included epidermal hyperplasia, skin edema and fibrosis. Furthermore, we also observed NO-dependent alleviation of the TPA-induced NK cell depletion in the ear tissues by heptaminol pretreatment. Together our results suggest that stimulation of NO generation from constitutive synthases may be primarily responsible for the reported antihyperplastic and NK cell-preserving effects of the ß-blockers, and that similar effects may be observed in other immunity normalizing compounds that also promote endothelial NO synthesis.

Nebivolol ameliorates asymmetric dimethylarginine-induced vascular response in rat aorta via ß3 adrenoceptor-mediated mechanism.

BACKGROUND: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, induces endothelial dysfunction. Nebivolol, a highly selective ß1-adrenergic receptor (AR) blocker, is the only beta-blocker known to induce vascular production of nitric oxide. OBJECTIVE: The present study was designed to evaluate the effect and mechanism of nebivolol on ADMA-induced vascular response in rat aorta in vitro. METHODS: In vitro, the effects of nebivolol and ADMA on resting tone or contraction induced by phenylephrine (PE, 10(-6 )mol/L) and relaxation induced by acetylcholine (Ach, 10(-10)-10(-5 )mol/L) were evaluated. RESULTS: ADMA in a concentration-dependent manner increased the resting and PE-induced tone and reduced Ach-induced relaxation. Nebivolol inhibited the ADMA-induced enhancements in tone and reversed the effects of ADMA on Ach-induced relaxation. These effects of nebivolol were blocked by selective ß3 receptor blocker cyanopindolol (1 µM), but not by selective ß2 receptor blocker butoxamine (50 µM). CONCLUSIONS: Nebivolol ameliorates the ADMA-induced vascular responses in rat aorta, at least in part, by mechanisms involving ß3 adrenoceptor.

Thyroid Hormone-Induced Differentiation of Astrocytes is Associated with Transcriptional Upregulation of ß-arrestin-1 and ß-adrenergic Receptor-Mediated Endosomal Signaling.

Thyroid hormones (TH) promote differentiation of astrocytes. We have previously reported that a downstream role ß-adrenergic receptor (ß-AR) system in such effects of TH. Although evidences indicate strong interaction between TH and the ß-ARs, the underlying mechanism is poorly understood. In the present study, we further explored the influence of TH on ß-AR signaling during the differentiation process. Unlike ß1-AR, binding of (125)I-pindolol to ß2-AR in cell membranes was significantly decreased at 2 h of exposure to TH which came back to control values after 24 h. The initial decrease in ß2-AR in membranes resulted in a concomitant increase in ß2-AR levels in the cytosol, suggesting that TH may induce endocytosis of the receptor. qRT-PCR as well as Western blot analysis demonstrated that unlike ß-adrenergic receptor kinase (ß-ARK)1 and ß-ARK2, the messenger RNA (mRNA) and protein levels of ß-arrestin-1 in the astrocyte cultures increased on exposure to TH. Knockdown of ß-arrestin gene suggested requirement of both ß-arrestin-1 and ß-arrestin-2 isoforms during endocytosis of ß2-AR, thereby facilitating cell differentiation. Endocytic inhibitors blocked the delayed but sustained activation of p-extracellular signal-regulated kinase (ERK) observed during cell differentiation. Observations suggest that TH upregulate ß-arrestin-1 in astrocytes to facilitate endocytosis of ß2-AR, required for endosomal ERK activation to drive the differentiation process.

Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol-Bound ß1-Adrenergic Receptor.

Comparisons between structures of the ß1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser(5.46), coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of ß1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both ß1AR and ß2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey ß1AR and an inverse agonist of human ß2AR. The structure of 7-methylcyanopindolol-bound ß1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound ß1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol-bound ß1AR and the hydroxyl group of Ser(5.46) is positioned 0.8 Å further from the ligand, with respect to the position of the Ser(5.46) side chain in cyanopindolol-bound ß1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.

Serotonergic 5-HT2A/2C receptors are involved in prolactin secretion in hyperestrogenic rats.

Serotonin (5-HT) has been shown to participate in prolactin secretion through a complex process resulting in both positive and negative effects. Estrogen has also been recognized as being involved in this serotonergic effect on prolactin release. Therefore, the aim of the present study was to assess whether estradiol modulates serotonergic involvement in prolactin secretion though 5-HT1A and/or 5-HT2A/2C receptors. Ovariectomized female Wistar rats, treated for 2 weeks with estrogen to induce a hyperprolactinemic status (hyperestrogenic rats) or with sunflower oil vehicle (hypoestrogenic rats), were injected daily with normal saline solution or 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), an 5-HT2A/2C receptor agonist, for 4 days. Other groups of ovariectomized animals received 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) or pindolol, an agonist and antagonist of the 5-HT1A receptor respectively, on the last day of treatment with estrogen or vehicle. Prolactin levels were compared among groups in each experiment under the distinct drug treatments. MK-212 was found to increase prolactin concentrations both in hyper- and hypoestrogenic females compared to controls (p<0.05). In contrast, prolactin levels remained similar to those of controls both in hyperestrogenic animals treated with 8-OH-DPAT and pindolol and in hypoestrogenic rats administered the same treatments. In conclusion, our findings indicate the involvement of 5-HT2A/2C receptors on prolactin release through serotonergic pathways in female animals, especially in hyperestrogenic states.

No evidence that MDMA-induced enhancement of emotional empathy is related to peripheral oxytocin levels or 5-HT1a receptor activation.

The present study aimed at investigating the effect of MDMA on measures of empathy and social interaction, and the roles of oxytocin and the 5-HT1A receptor in these effects. The design was placebo-controlled within-subject with 4 treatment conditions: MDMA (75 mg), with or without pindolol (20 mg), oxytocin nasal spray (40 IU+16 IU) or placebo. Participants were 20 healthy poly-drug MDMA users, aged between 18-26 years. Cognitive and emotional empathy were assessed by means of the Reading the Mind in the Eyes Test and the Multifaceted Empathy Test. Social interaction, defined as trust and reciprocity, was assessed by means of a Trust Game and a Social Ball Tossing Game. Results showed that MDMA selectively affected emotional empathy and left cognitive empathy, trust and reciprocity unaffected. When combined with pindolol, these effects remained unchanged. Oxytocin did not affect measures of empathy and social interaction. Changes in emotional empathy were not related to oxytocin plasma levels. It was concluded that MDMA (75 mg) selectively enhances emotional empathy in humans. While the underlying neurobiological mechanism is still unknown, it is suggested that peripheral oxytocin does not seem to be the main actor in this; potential candidates are the serotonin 2A and the vasopressin 1A receptors. Trial registration: MDMA & PSB NTR 2636.

Intermolecular complexation of low-molecular-weight succinoglycans directs solubility enhancement of pindolol.

The low-molecular-weight succinoglycans isolated from Sinorhizobium meliloti are repeating octasaccharide units consisting of monomers, dimers, and trimers. Pindolol is a beta-blocker used to treat cardiovascular disorders. We investigated the formation of complexes between pindolol and low-molecular-weight succinoglycan monomers (SGs). Even though SGs have a linear structure, the solubility of pindolol in the presence of SGs was increased up to 7-fold compared with methyl-ß-cyclodextrin reported as the best solubilizer of pindolol. Complexation of SGs with pindolol was confirmed by nuclear magnetic resonance, Fourier-transform infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy. Formation constants of complexes were determined from phase solubility diagrams. Conformation of complex was suggested based on a molecular docking study. The present study indicated that formation of pindolol/SGs complexes not only resulted in increased pindolol solubility but also could be useful for improving its clinical application as it did not affect cell viability.

Case study 6. Transporter case studies: in vitro solutions for translatable outcomes.

Assessing the interactions of a new drug candidate with transporters, either as a substrate or as an inhibitor, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to eleven for the EMA as of 2013. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to really appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the best tools for the job and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in transporter kinetics to the reader.

5-HT1B receptor modulation of the serotonin transporter in vivo: studies using KO mice.

The serotonin transporter (SERT) controls the strength and duration of serotonergic neurotransmission by the high-affinity uptake of serotonin (5-HT) from extracellular fluid. SERT is a key target for many psychotherapeutic and abused drugs, therefore understanding how SERT activity and expression are regulated is of fundamental importance. A growing literature suggests that SERT activity is under regulatory control of the 5-HT1B autoreceptor. The present studies made use of mice with a constitutive reduction (5-HT1B+/-) or knockout of 5-HT1B receptors (5-HT1B-/-), as well as mice with a constitutive knockout of SERT (SERT-/-) to further explore the relationship between SERT activity and 5-HT1B receptor expression. High-speed chronoamperometry was used to measure clearance of 5-HT from CA3 region of hippocampus in vivo. Serotonin clearance rate, over a range of 5-HT concentrations, did not differ among 5-HT1B receptor genotypes, nor did [(3)H]cyanoimipramine binding to SERT in this brain region, suggesting that SERT activity is not affected by constitutive reduction or loss of 5-HT1B receptors; alternatively, it might be that other transport mechanisms for 5-HT compensate for loss of 5-HT1B receptors. Consistent with previous reports, we found that the 5-HT1B receptor antagonist, cyanopindolol, inhibited 5-HT clearance in wild-type mice. However, this effect of cyanopindolol was lost in 5-HT1B-/- mice and diminished in 5-HT1B+/- mice, indicating that the 5-HT1B receptor is necessary for cyanopindolol to inhibit 5-HT clearance. Likewise, cyanopindolol was without effect on 5-HT clearance in SERT-/- mice, demonstrating a requirement for the presence of both SERT and 5-HT1B receptors in order for cyanopindolol to inhibit 5-HT clearance in CA3 region of hippocampus. Our findings are consistent with SERT being under the regulatory control of 5-HT1B autoreceptors. Future studies to identify signaling pathways involved may help elucidate novel therapeutic targets for the treatment of psychiatric disorders, particularly those linked to gene variants of the 5-HT1B receptor.

Both acute and subchronic treatments with pindolol, a 5-HT(1A) and ß1 and ß2 adrenoceptor antagonist, elevate regional serotonin synthesis in the rat brain: an autoradiographic study.

Antidepressant treatments, including those that increase serotonin (5-HT) neurotransmission, require several weeks or months until the onset of the therapeutic effect in depressed patients. The negative feedback on 5-HT transmission exhibited by the 5-HT(1A) and 5-HT(1B) autoreceptors has been postulated as a possible delaying factor. The aim of the present study was to assess the effect of the acute and subchronic treatment with pindolol, a 5-HT(1A/1B,) ß1 and ß2 adrenoceptor antagonist, on 5-HT synthesis, one of the key parameters of 5-HT neurotransmission. Male Sprague-Dawley (SPD) rats (180-220 g) were treated with pindolol or an adequate volume of saline, administered either acutely (15 mg/kg i.p.; SPD-AC-SAL, SPD-AC-TR) or subchronically (15 mg/kg day i.p. for 7 days; SPD-SUBCHR-SAL, SPD-SUBCHR-TR). Thirty minutes following the single i.p. injection (acute experiment) or at the 8th day following the commencement of the subchronic treatment (subchronic experiment), 5-HT synthesis was measured using α-[¹4C]methyl-L-tryptophan autoradiography. The analysis of variance (ANOVA), followed by the Benjamini-Hochberg correction for multiple comparisons, revealed: (1) a significant increase of 5-HT synthesis in the SPD-AC-TR rats, relative to the SPD-AC-SAL rats in all brain regions examined except the substantia nigra--pars reticularis, dorsal subiculum, inferior olive, raphe magnus and raphe obscurus and (2) a significant increase of 5-HT synthesis in the SPD-SUBCHR-TR rats, relative to the SPD-SUBCHR-SAL rats in all brain regions except the median raphe, hypothalamus and raphe pontine. On the basis of these results, we hypothesized that the antagonism of the 5-HT(1A/1B) receptors prevents the negative feedback mediated by these receptors on 5-HT synthesis, resulting in a persistent increase of 5-HT synthesis. The results accord with clinical reports on the utility of pindolol in the augmentation of antidepressant treatment.