A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR-G protein interactions.

G protein-coupled receptors (GPCRs) are currently the target of more than 30% of the marketed medicines. However, there is an important medical need for ligands with improved pharmacological activities on validated drug targets. Moreover, most of these ligands remain poorly characterized, notably because of a lack of pharmacological tools. Thus, there is an important demand for innovative assays that can detect and drive the design of compounds with novel or improved pharmacological properties. In particular, a functional and screening-compatible GPCR-G protein interaction assay is still unavailable. Here, we report on a nanoluciferase-based complementation technique to detect ligands that promote a GPCR-G protein interaction. We demonstrate that our system can be used to profile compounds with regard to the G proteins they activate through a given GPCR. Furthermore, we established a proof of applicability of screening for distinct G proteins on dopamine receptor D2 whose differential coupling to Gαi/o family members has been extensively studied. In a D2-Gαi1versus D2-Gαo screening, we retrieved five agonists that are currently being used in antiparkinsonian medications. We determined that in this assay, piribedil and pergolide are full agonists for the recruitment of Gαi1 but are partial agonists for Gαo, that the agonist activity of ropinirole is biased in favor of Gαi1 recruitment, and that the agonist activity of apomorphine is biased for Gαo We propose that this newly developed assay could be used to develop molecules that selectively modulate a particular G protein pathway.

Determination of pergolide in horse plasma by UPLC-MS/MS for pharmacokinetic applications.

Pergolide, an ergot-derived dopamine D2 receptor agonist, is used extensively as an orally administered treatment for pituitary pars intermedia dysfunction (PPID) in horses. One of the barriers associated with pergolide determinations in plasma for pharmacokinetic applications has been the technically demanding requirement for sensitivity. The objective of our work was to develop a simple assay for the determination of pergolide in plasma and demonstrate its potential application in the study of pergolide pharmacokinetics (PK) in horses. A UPLC-MS/MS assay was developed with a simple sample preparation involving methanol protein precipitation and injection of supernatant. The assay was applied to samples from a horse dosed with 10mg pergolide (as the mesylate salt) by nasogastric intubation. Plasma samples were collected over a 48h period. The assay demonstrated performance sufficient to enable application to low level PK studies. Within-batch precision and accuracy were within acceptance criteria; precision was less than 10% RSD (n=5) and accuracy was -7.3% at 0.014ng/mL, the lower limit of quantification was 0.006ng/mL and the method detection limit was 0.002ng/mL. In the treated horse, Cmax was 0.40ng/mL and the assay easily allowed determination of plasma levels in the elimination phase to 48h. In conclusion, this assay using UPLC-MS/MS and methanol protein precipitation easily meets the challenging demands of pergolide analyses in plasma.

Multitemperature stability and degradation characteristics of pergolide mesylate oral liquid.

The stability of pergolide mesylate in an oral aqueous liquid was studied. Stability and solubility data were used to determine the degradation characteristics of the drug in this formulation. Samples were stored in the dark at 35°C, 45°C, and 60°C. At 1, 2, 4, 8, 12, and 16 weeks, samples were removed and stored in a -80°C freezer for high performance liquid chromatography (HPLC) assay at a later date. The initial drug concentration of 0.30 mg/mL was determined by assay after storage at -80°C. A solubility of 6.9 mg/mL was found for pergolide mesylate in the oral liquid at room temperature with a relative standard deviation (RSD) of 4.0%. The degradation process is considered first-order at 25°C and 35°C. At higher temperatures (45°C and 60°C), a color change and curvature at the latter time points in degradation profiles are ascribed to the presence of methylcellulose. The activation energy calculated for degradation of pergolide mesylate in the oral liquid was 21.3 kcal/mol. The time to reach 90% potency (t90) values were calculated to be 43 days and 3 days, respectively, for storage at 25°C and 35°C. Drug concentrations up to ~6 mg/mL can be maintained as a solution at room temperature with this formulation.

Effects of compounding and storage conditions on stability of pergolide mesylate.

OBJECTIVE: To determine the effects of temperature and light over a 35-day period on stability of pergolide mesylate after compounding in an aqueous vehicle. DESIGN: Evaluation study. PROCEDURES: Pergolide was compounded into a formulation with a final target concentration of 1 mg/mL. Aliquots of the formulation were then stored at -20 degrees, 8 degrees, 25 degrees, or 37 degrees C without exposure to light or at 25 degrees C with exposure to light for 35 days. Samples were assayed in triplicate by means of high-pressure liquid chromatography immediately after compounding and after 1, 7, 14, 21, and 35 days of storage. RESULTS: Mean+/-SD concentration of pergolide in the formulation immediately after compounding was 1.05+/-0.086 mg/mL. Samples exposed to light while stored at 25 degrees C had undergone excessive degradation by day 14, samples stored at 37 degrees C had undergone excessive degradation by day 21, and samples stored at 25 degrees C without exposure to light had undergone excessive degradation by day 35. The decrease in expected concentration corresponded with the appearance of degradation peaks in chromatograms and with a change in color of the formulation. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that pergolide mesylate was unstable after compounding in an aqueous vehicle and that storage conditions had an effect on stability of the compounded formulation. Compounded pergolide formulations in aqueous vehicles should be stored in a dark container, protected from light, and refrigerated and should not be used >30 days after produced. Formulations that have undergone a color change should be considered unstable and discarded.

A ligand-based approach to mining the chemogenomic space of drugs.

The practical implementation and validation of a ligand-based approach to mining the chemogenomic space of drugs is presented and applied to the in silico target profiling of 767 drugs against 684 targets of therapeutic relevance. The results reveal that drugs targeting aminergic G protein-coupled receptors (GPCRs) show the most promiscuous pharmacological profiles. The detection of cross-pharmacologies between aminergic GPCRs and the opioid, sigma, NMDA, and 5-HT3 receptors aggravate the potential promiscuity of those drugs, predominantly including analgesics, antidepressants, and antipsychotics.

Characterization of the molecular fragment that is responsible for agonism of pergolide at serotonin 5-Hydroxytryptamine2B and 5-Hydroxytryptamine2A receptors.

Cardiac-valve regurgitation observed in Parkinson patients treated with the ergoline dopamine receptor agonist 8beta-methylthiomethyl-6-propylergoline (pergolide) has been associated with the agonist efficacy of the drug at 5-hydroxytryptamine(2B) (5-HT(2B)) receptors. 5-HT(2A) receptors may also play a role in pergolide-induced cardiac-valve regurgitation. We studied the pharmacological profile of pergolide and eight derivatives in porcine vascular rings endowed with 5-HT(2B) and 5-HT(2A) receptors to detect the molecular fragment of the pergolide molecule that may be responsible for agonism at these receptors. Pergolide derivatives showed a different substitution pattern at N(6), and the side chain at C(8) was modified by replacement of the sulfur against an oxygen atom. We demonstrate that the potent agonism of pergolide both at 5-HT(2B) and 5-HT(2A) receptors is retained when the N(6) propyl substituent is replaced by ethyl. However, agonism can be converted into antagonism if N(6) propyl is replaced by methyl. The N(6)-unsubstituted derivative was a low efficacy 5-HT(2B) receptor partial agonist and a 5-HT(2A) receptor antagonist. This pharmacological pattern was also applicable for pergolide congeners with an oxygen in the side chain at C(8). 6-Methylpergolide retained agonist efficacy and potency compared with pergolide at human (h) D(2LONG(L)) and hD(2SHORT(S)) receptors as determined by guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Based on the ability of pergolide to produce potent agonism at 5-HT(2B) receptors and the failure of 6-methylpergolide to act as an agonist but as a potent antagonist, we conclude that the N(6) propyl substituent of pergolide is crucial for 5-HT(2B) receptor agonism and thus a determinant of valvular regurgitation.

Anti-fibrillogenic and fibril-destabilizing activities of anti-Parkinsonian agents for alpha-synuclein fibrils in vitro.

The aggregation of alpha-synuclein (alphaS) in the brain has been implicated as a critical step in the development of Lewy body diseases (LBD) and multiple system atrophy (MSA). Among the antioxidant strategies proposed, increasing evidence points to the possibility of achieving neuroprotection by dopamine agonists, as well as monoamine oxidase B inhibitors. We showed previously that the anti-Parkinsonian agents dose-dependently inhibited beta-amyloid fibrils (fAbeta)(1-40) and fAbeta(1-42) formation as well as destabilized preformed fAbetas. Using fluorescence spectroscopy with thioflavin S, electron microscopy, and atomic force microscopy, we examined the effects of anti-Parkinsonian agents, selegiline, dopamine, pergolide, bromocriptine, and trihexyphenidyl on the formation of alphaS fibrils (falphaS) and on preformed falphaS. All molecules except for trihexyphenidyl, dose-dependently inhibited the formation of falphaS. Moreover, these molecules dose-dependently destabilized preformed falphaS. The overall activity of the molecules examined was in the order of: selegiline = dopamine > pergolide > bromocriptine. These agents and other compounds related structurally could be key molecules for the development of therapeutics for LBD and MSA.

Improved photostability indicating ion-pair chromatography method for pergolide analysis in tablets and in the presence of cyclodextrins.

Pergolide (PG) a semi-synthetic ergot alkaloid derivative used mainly for the treatment of Parkinson's disease is known to be a photosensitive drug substance. The major photodegradation products are PG sulphoxide (SX) and PG sulphone (SN), which are also the main impurities of the bulk drug substance. It is widely metabolized to more than 10 metabolites including SX and SN. In this work an improved photostability indicating ion-pair chromatography method for PG mesilate was developed. The method can be applied in the determination of PG and impurities in aqueous solutions and in tablets for routine analysis. This new method is appropriate for the quantitative determination of PG in the presence of its impurities and photodegradation products and can also be used for PG complexes with cyclodextrins (commonly used as photostabilizing agents). Furthermore it is suitable for the quantitation of its impurities and its thermal or photo-induced decomposition products. Separation was achieved on a ThermoQuest C(18) BDS column and Sodium octanosulphonate was used as ion-pairing agent. Analysis was performed at 223 nm. Validation parameters included: specificity, linearity, precision and accuracy, limit of quantitation and suitability. The method was found to be specific and linear for PG, as well as for SX and, SN impurities. The recovery was 100.83+/-0.46% for PG, 99.86+/-0.33% for SX and 99.77+/-1.84% for SN. Finally the photodegradation profile of PG mesilate was studied in different initial sample concentration. The obtained result revealed that: PG photolysis is catalyzed by its degradation products and that decrease of initial sample concentration reduces the rate of PG photoinduced degradation.

Pleuropulmonary fibrosis after long-term treatment with the dopamine agonist pergolide for Parkinson Disease.

Dopamine agonists are increasingly used in the treatment of Parkinson disease, but they may cause serious adverse effects. In December 1983, symptoms of Parkinson disease developed in a 55-year-old man with no history of pulmonary disease, smoking, or asbestos exposure. He began treatment with dopamine agonists bromocriptine mesylate (in 1984) and pergolide mesylate (in 1989). In late 2000, pulmonary symptoms developed. Chest radiographs and computed tomographic findings showed a mass in the right upper lobe and effusion. A biopsy specimen showed pleural and parenchymal fibrosis. This syndrome resolved after cessation of pergolide therapy and a switch to pramipexole dihydrochloride. This case draws attention to the association of long-term ergot dopamine agonist therapy with pleuropulmonary fibrosis, which can develop as late as 11 years after the initiation of therapy. We also review evidence that the risk of this complication is substantially lower with the newer nonergot dopamine agonists.

Severe multivalvular heart disease: a new complication of the ergot derivative dopamine agonists.

We report on 4 new cases of valvular heart disease in Parkinson's disease patients treated with the ergot derivative dopamine agonists pergolide and cabergoline. Noninflammatory fibrotic degeneration of cardiac valves has been reported to occur in patients with carcinoid syndrome and to occasionally complicate therapies with the anti-migraine ergot alkaloid ergotamine and methysergide and with the appetite suppressants fenfluramine and dexfenfluramine. In these cases, the pathogenesis is suspected to involve serotonin-mediated abnormal fibrogenesis by means of the 5-HT2B receptors, which are expressed in the fibroblasts of heart valves. Based on strikingly similar echocardiographic and histopathological features, we strongly suspect that ergot-derived dopamine agonists may cause a valvular heart disease nearly identical to that seen in those conditions. These cases add to a rapidly growing and worrying list of similar published reports, suggesting that we may well be facing a novel, yet unrecognized, complication of this class of agents, which are widely used not only in Parkinson's disease but also in restless legs syndrome and various common endocrine dysfunctions. Therefore, until more is known about the true prevalence of this side effect, we propose that an assessment of cardiac function be performed before and in the course of a long-term therapy with ergot derivative dopamine agonists.

Pergolide, terguride and N,N'-spacer-linked oligomers of both interact with 5-HT2A receptors of rat tail artery.

Pergolide, terguride and N,N'-spacer-linked oligomers of both have been tested for their ability to interact with 5 hydroxytryptamine(HT)2A receptors of rat tail artery. Pergolide was a potent partial agonist (pEC50 7.5, Emax 55 %) and antagonized 5-HT-induced contractions (pKp 7.2). Pergolide dimer 3 with a p-xylene spacer between the indole nitrogens (N-1) displayed somewhat lower agonist potency than pergolide (pEC50 7.0, Emax 55 %, pKp 6.6). The contractile responses to pergolide and dimer 3 were antagonized by the 5-HT2A receptor antagonist ketanserin (pA2 9.4, 9.1). In contrast to pergolide dimer 3, pergolide dimers 5 and 9 with an alkyl and an aralkyl spacer between the piperidine nitrogens (N-6) lacked agonism and displayed low affinity at 5-HT2A receptors (pA2 < 5.5). Terguride behaved as an insurmountable antagonist of 5-HT (pA2 8.4). Oligomers of terguride showed 5 to 50-fold lower affinity. It is concluded that pergolide and terguride show a high affinity for 5-HT2A receptors, but dimerization (oligomerization) of both drugs fails to increase affinity.

Pergolide mesylate form II.

A new polymorph of pergolide mesylate or 8beta-[(methylsulfanyl)methyl]-6-propylergoline methanesulfonate, C(19)H(27)N(2)S(+).CH(3)SO(3)(-), is reported. Pergolide mesylate form II crystallizes in the trigonal system, which is unique for ergot derivatives. Although the hydrogen-bond system in form II differs completely from that in form I, the conformation of the pergolide moiety in various related structures is very similar.

The in vitro transport of pergolide from surfactant-based elastic vesicles through human skin: a suggested mechanism of action.

This paper reports the in vitro transport of pergolide from L-595-PEG-8-L elastic vesicle formulations. Several aspects of vesicular delivery were studied in order to elucidate the possible mechanisms of action and to establish the optimal conditions and drug candidates for usage with L-595-PEG-8-L elastic vesicles. All studies were performed using human skin and flow-through Franz diffusion cells. Pergolide was chosen as model drug. The findings show that there was a strong correlation between the drug incorporation to saturated levels and the drug transport, both of which were influenced by the pH of the drug-vesicular system. The optimal pH was found to be 5.0, giving the highest drug incorporation as well as the highest drug transport. Non-occlusive co-treatment with elastic vesicles improved the skin delivery of pergolide compared to the non-occlusive buffer control by more than 2-fold. However, non-occlusive pre-treatment of skin with empty vesicles did not enhance drug transport. Occlusion improved drug transport from both elastic vesicle as well as buffer solutions due to the fact that water is an excellent penetration enhancer for pergolide. However, in contrast to non-occlusive application, the action of the elastic vesicles themselves was diminished, as occlusive treatments with elastic vesicles showed a lower flux compared to occlusive treatment with the buffer control. Hence, the highest pergolide skin permeation in this study was obtained from an occluded saturated buffer solution, giving a steady-state flux of 137.9 ng/h cm(-2). The volume of application did not have any effect on the drug transport. In conclusion, these results showed no evidence that a penetration enhancing effect is the main mechanism of action. The pH of the drug-vesicular system is an important factor to consider when optimising elastic vesicle delivery systems. Occlusion reduces the actions of elastic vesicles, but could increase the pergolide transport since water is a good penetration enhancer for this particular drug. Based on the results obtained, a mechanism of action for the elastic vesicles was proposed.

CoMFA-based prediction of agonist affinities at recombinant wild type versus serine to alanine point mutated D2 dopamine receptors.

Agonist affinity changes dramatically as a result of serine to alanine mutations (S193A, S194A, and S197A) within the fifth transmembrane region of D2 dopamine receptors and other receptors for monoamine neurotransmitters. However, agonist 2D-structure does not predict which drugs will be sensitive to which point mutations. Modeling drug-receptor interactions at the 3D level offers considerably more promise in this regard. In particular, a comparison of the same test set of agonists across receptors differing minimally (point mutations) offers promise to enhance the understanding of the structural bases for drug-receptor interactions. We have previously shown that comparative molecular field analysis (CoMFA) can be applied to comparisons of affinity at recombinant D1 and D2 dopamine receptors for the same set of agonists, a differential QSAR. Here, we predicted agonist K(L) for the same set of agonists at wild type D2 vs S193A, S194A, and S197A receptors using CoMFA. Each model used bromocriptine as the template. ln(1/K(L)) values for the low-affinity agonist binding conformation at recombinant wild type and mutant D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA of the 16 aligned agonist structures. The resulting CoMFA models yielded cross-validated R(2) (q(2)) values ranging from 0.835 to 0.864 and simple R(2) values ranging from 0.999 to 1.000. Predictions of test compound affinities at WT and each mutant receptor were close to measured affinity values. This finding confirmed the predictive ability of the models and their differences from one another. The results strongly support the idea that CoMFA models of the same training set of compounds applied to WT vs mutant receptors can accurately predict differences in drug affinity at each. Furthermore, in a "proof of principle", two different templates were used to derive the CoMFA model for the WT and S193A mutant receptors. Pergolide was chosen as an alternate template because it showed a significant increase in affinity as a result of the S193A mutation. In this instance both the bromocriptine- and pergolide-based CoMFA models were similar to one another but different from those for the WT receptor using bromocriptine- or pergolide- as templates. The pergolide-based S193A model was more strikingly different from that of the WT receptor than was the bromocriptine-based S193A model. This suggests that a "dual-template" approach to differential CoMFA may have special value in elucidating key differences across related receptor types and in determining important elements of the drug-receptor interaction.