Concise Synthesis of Both Enantiomers of Pilocarpine.

Furan-2-carboxylic acid was used as a starting material for the synthesis of dehydro-homopilopic acid. Esterification, hydrogenation and enzymatic hydrolysis followed by the reduction of Weinreb amides and a single-step attachment of a 1-methyl-imidazole residue allowed for the concise synthesis of both enantiomers of pilocarpine.

Gastrodin attenuates lithium-pilocarpine-induced epilepsy by activating AMPK-mediated PPARα in a juvenile rat model.

Gastrodin has shown the potential as an anticonvulsant. Epilepsy is a neurological disease with significant effects in children. In the current study, the therapeutic potential of gastrodin in handling pediatric epilepsy was explored by focusing on the AMPK/PPARα pathway. Three-week-old Sprague-Dawley rats were subjected to lithium-pilocarpine method to induce epileptic symptoms and then administrated with gastrodin. The effects of gastrodin on rats were first assessed using electroencephalogram (EEG) recording, Racine classification, Morris water maze test, and histological staining. The levels of BDNF and NGF, and the activity of AMPK/PPARα were measured. Based on the results of EEG, behavior analyses, and histological staining, epileptic symptoms were significantly alleviated by gastrodin. Moreover, the administration of gastrodin also suppressed the levels of BDNF and NGF, and activated the AMPK/PPARα pathway. In conclusion, our results demonstrated that gastrodin contributed to the alleviation of pediatric epilepsy by activating AMPK/PPARα signaling transduction.

Gellan gum and its methacrylated derivatives as in situ gelling mucoadhesive formulations of pilocarpine: In vitro and in vivo studies.

Gellan gum was chemically modified by the reaction with methacrylic anhydride to produce derivatives with 6, 14 and 49% methacrylation. The structure and substitution degrees of these derivatives were confirmed by 1H NMR- and FTIR-spectroscopy. These derivatives are more hydrophobic compared to pristine gellan and form turbid solutions in water. In vitro study performed with formulations of sodium fluorescein containing gellan gum and its methacrylated derivatives indicated that methacrylation enhances their retention on bovine conjunctival mucosa. In vivo experiments with the formulations of pilocarpine hydrochloride containing gellan gum and methacrylated derivatives have demonstrated that all polymers enhance the drug effect significantly, but best performance is observed for the polysaccharide with 6% methacrylation.

Solid-state epimerisation and disproportionation of pilocarpine HCl: Why we need a 5-stage approach to validate melting point measurements for heat-sensitive drugs.

Melting points for new drugs are reported in regulatory documents, e.g. investigational brochures, and frequently in published research; however, the authors do not typically consider that heat-induced degradation can affect the melting point measurement. Applying a single heating rate is not adequate, and thus many melting points in the literature and regulatory documentation are not valid. Our aim was to validate a five-stage approach for the melting point measurement of heat-sensitive drugs. These stages are; (1) observe melting; (2) record mass loss; (3) measure melting points at different heating rates; (4) characterise degradation and (5) test for potential isomerisation. Applying this approach to pilocarpine HCl illustrated the sensitivity of a melting point to thermal degradation. Due to salt disproportionation & loss of HCl gas, pilocarpine's melting point decreased by 14 °C when the heating rate was lowered from 20 to 1 °C/min. Epimerization occurred before melting was reached. Increasing the heating rate diminished disproportionation; however, this did not remove epimerization. Thus, the melting point of pilocarpine HCl of 205.5 ± 0.4 °C measured at 20 °C/min represents the melt of a racemic mixture containing inactive isopilocarpine. Heating above the melting point accelerated degradation, a rate of 5 °C/min recovered just 38 ± 1% of pilocarpine. Such data predicted a shelf-life of 6.6 years. Pilocarpine successfully validated the multistage approach by providing new knowledge concerning its thermal stability. Our 5-stage approach must be applied to all new drugs especially if their formulation requires heat. For example, thermal stability is an infrequently considered pre-requisite in the emerging field of 3D printing.

Dendritic Effects of Injectable Biodegradable Thermogels on Pharmacotherapy of Inflammatory Glaucoma-Associated Degradation of Extracellular Matrix.

The development of advanced drug delivery systems with extensively sustained release and multiple functions is highly imperative for effective attenuation of the degradation of ocular extracellular matrix that is associated with inflammatory glaucoma. Here, the generation of amine-terminated polyamidoamine dendrimers in an injectable biodegradable thermogel is demonstrated to be important for achieving prolonged drug release profiles and potent anti-inflammatory effects. Among various generations (Gx, x = 0, 1, 3, 5), third-generation G3 is proved as the most effective material for optimizing the synergistic effects of gelatin and poly(N-isopropylacrylamide) and generating a thermogel with the highest biodegradation resistance, the best drug encapsulation/extended-release performance, and the best ability to reduce the elevated expression of inflammatory molecules. A pharmacotherapy based on intracameral injection of thermogels coloaded with pilocarpine and ascorbic acid results in effective alleviation of progressive glaucoma owing to the anti-inflammatory activity and long-acting drug release (above a therapeutic level of 10 µg mL-1 over 80 days) of thermogels, which simultaneously suppress inflammation and stimulate regeneration of stromal collagen and retinal laminin. These findings on the dendritic effects of rationally designed injectable biomaterials with potent anti-inflammatory effects and controlled drug release demonstrate great promise of their use for pharmacological treatment of progressive glaucoma.

Cubic and hexagonal liquid crystal gels for ocular delivery with enhanced effect of pilocarpine nitrate on anti-glaucoma treatment.

The objective of this work was to investigate phytantriol-based liquid crystal (LC) gels including cubic (Q2) and hexagonal (H2) phase for ocular delivery of pilocarpine nitrate (PN) to treat glaucoma. The gels were produced by a vortex method and confirmed by crossed polarized light microscopy, small-angle X-ray scattering, and rheological measurements. Moreover, the release behaviors and permeation results of PN from the gels were estimated using in vitro studies. Finally, the anti-glaucoma effect of LC gels was evaluated by in vivo animal experiments. The inner structure of the gels was Pn3m-type Q2 and H2 phase, and both of them showed pseudoplastic fluid properties based on characterization techniques. In vitro release profiles suggested that PN could be sustainably released from LC gels within 48 h. Compared with eye drops, Q2 and H2 gel produces a 5.25-fold and 6.23-fold increase in the Papp value (p < .05), respectively, leading to a significant enhancement of corneal penetration. Furthermore, a good biocompatibility and longer residence time on precorneal for LC gels confirmed by in vivo animal experiment. Pharmacokinetic studies showed that LC gels could maintain PN concentration in aqueous humor for at least 12 h after administration and remarkably improve the bioavailability of drug. Additionally, in vivo pharmacodynamics studies indicated that LC gels had a more significant intraocular pressure-lowering and miotic effect compared to eye drops. These research findings hinted that LC gels would be a promising pharmaceutical strategy for ocular application to enhance the efficacy of anti-glaucoma.

Buccal adhesive chitosan conjugate comprising pilocarpine for xerostomia.

BACKGROUND: Xerostomia is caused by different factors such as side effects of medication, radiotherapy by head and neck cancer as well as Sjögren syndrome. AIM: The goal was to synthesize novel preactivated chitosan conjugates and to design adhesive dosage forms comprising sialagogue pilocarpine. METHODS: Unmodified chitosan (CH) was covalently linked to sulfhydryl possessing mercaptonicotinic acid (MNA) via amide bond formation. In a second step, preactivation occurred via disulfide bond establishment between sulfhydryl linked chitosan and preactivation ligand MNA. Mucoadhesive and mucoprotective properties were scrutinized on buccal mucosa. Safety assessment was performed on head and neck squamous cells. Histology assay was conducted on buccal tissue. Pilocarpine was scrutinized in terms of controlled release behavior. RESULTS: Novel preactivated CH was successfully synthesized and considered as not harmful to the cells at all. Furthermore, mucoadhesion was 1.3-fold improved in the presence of preactivated chitosan as compared to respective unmodified one. Pilocarpine exhibited a 3.1-fold controlled release in presence of novel synthesized chitosan as in comparison to unmodified CH. CONCLUSION: The novelty of this promising polymeric carrier lies in the synthesis procedure leading to a pronounced mucoadhesive, mucoprotecting and controlled release encouraging dosage form in the management of xerostomia.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Self-assembled hexagonal liquid crystalline gels as novel ocular formulation with enhanced topical delivery of pilocarpine nitrate.

The aim of this paper was to develop hexagonal liquid crystalline (HII) gels that can be used as a novel ocular delivery system for pilocarpine nitrate (PN). HII gels were prepared by a vortex method using phytantriol/triglyceride/water (71.15: 3.85: 26, w/w) ternary system. The gels were characterized by crossed polarized light microscopy, small-angle X-ray scattering, differential scanning calorimetry and rheology. And, in vitro drug release behavior and ex vivo corneal permeation were investigated. Finally, preocular residence time evaluation, eye irritation test, histological examination and miotic tests were studied in vivo and compared with carbopol gel. Based on various characterization techniques, the inner structure of the gels were HII mesophase and exhibited a pseudoplastic fluid behaviour. In vitro release results revealed that PN could be released continuously from HII gel over a period of 24 h. The ex vivo apparent permeability coefficient of HII gel was 3.15-fold (P < 0.01) higher than that of the Carbopol gel. Compared with Carbopol gel, HII gel displayed longer residence time on the eyeballs surface using fluorescent labeling technology. Furthermore, the HII gel caused no ocular irritation was estimated by corneal hydration levels, Draize test and histological inspection. Additionally, in vivo miotic study showed that HII gel had a remarkably long-lasting decrease in the pupil diameter of rabbits. In conclusion, HII gels would be a promising sustained-release formulation for ocular drug delivery.

Effect of cellulose nanocrystals on the performance of drug loaded in situ gelling thermo-responsive ophthalmic formulations.

Triblock poloxamer copolymer (PM) has been extensively utilized to deliver various ophthalmic pharmaceutical compounds. The aim of efficient ophthalmic drug delivery strategy is to attain the longer precorneal resident time and good bioavailability of drugs. In this pursuit, the influence of cellulose nanocrystals (CNC) on the in situ gelation behavior of PM and in vitro release of pilocarpine hydrochloride from the nanocomposites formulations was studied. The critical concentration of gelation of PM being 18% (wt/v) was dropped to 16.6% (wt/v) by the addition of a very low percentage of CNC. The reinforcing nature of CNC via H-bonding in the in situ nanocomposite gel also led to an increase in gel strength along with the sustained release of loaded drugs when compared with the pure PM gel. All formulations revealed that the drug release mechanism is controlled by the Fickian diffusion. Thus, the CNC has a significant effect on the gelation behavior, gel strength, and drug release kinetics of PM-CNC formulations.

Effect of deacetylation degree on controlled pilocarpine release from injectable chitosan-g-poly(N-isopropylacrylamide) carriers.

Development of biodegradable thermogels as intracameral injectable carriers for ocular delivery of antiglaucoma medications can provide a better treatment modality with low dosing frequency than eye drop formulations. For the first time, this study investigates the effect of deacetylation degree (DD) of the polysaccharide component in chitosan-g-poly(N-isopropylacrylamide) (CN) carriers on controlled release of pilocarpine in the management of glaucoma. Our results showed that increasing the chitosan DD from 60.7% to 98.5% leads to enhanced biodegradation resistance of carrier and prolonged release profile of the drug. Significant DNA damage and caspase-3 activation could be detected in lens epithelial cell cultures exposed to CN made from highly deacetylated polysaccharides, indicating apoptosis-related cytotoxicity due to relatively high positive charge density of the graft copolymers. Postoperative outcomes demonstrated that long-term therapeutic efficacy in glaucomatous rabbits is governed by intraocular pressure changes in response to intracamerally administered pilocarpine-loaded CN, strongly suggesting the usefulness of deacetylation in this injectable drug delivery carrier.

Development and validation of a rapid RP-HPLC-DAD analysis method for the quantification of pilocarpine in Pilocarpus microphyllus (Rutaceae).

A method using high performance liquid chromatography with diode array detector (HPLC-DAD) for identification and quantification of pilocarpine in the extract of Pilocarpus microphyllus, popularly known as jaborandi. The analysis was conducted using RP-18 column (250 mm × 4.6 mm x 5 µm id) and a buffer solution composed of acidified water, phosphoric acid and triethylamine and methanol as a mobile phase in a flow rate of 1.0 mL/min and detection at 215  nm at 25 °C. Excellent linearity with r2 equal to 0.9999 was obtained. The recovery percentage was very satisfactory with values within the specifications. It is correct to affirm that the method has optimal intracurrent and intercurrent precision values with relative standard deviations of 0.1852% and 0.1932%, respectively. The robustness of the method, assessed through the Youden test, showed no significant influence of any of the evaluated parameters. In general, the method proved to be suitable for the intended purpose.

Exploring the structure characteristics and major channels of cytochrome P450 2A6, 2A13, and 2E1 with pilocarpine.

The majority of cytochromes P450 play a critical role in metabolism of endogenous and exogenous substrates, some of its products are carcinogens. Therefore, inhibition of P450 enzymes activity can promote the detoxification and elimination of chemical carcinogens. In this study, molecular dynamics (MD) simulations and adaptive steered molecular dynamics (ASMD) simulations were performed to explore the structure features and channel dynamics of three P450 isoforms 2A6, 2A13, and 2E1 bound with the common inhibitor pilocarpine. The binding free energy results combined with the PMF calculations give a reasonable ranking of binding affinity, which are consistent with the experimental data. Our results uncover how a sequence divergence of different CYP2 enzymes causes individual variations in major channel selections. On the basis of channel bottleneck and energy decomposition analysis, we propose a gating mechanism of their respective major channels in three enzymes, which may be attributed to a reversal of Phe209 in CYP2A6/2A13, as well as the rotation of Phe116 and Phe298 in CYP2E1. The hydrophobic residues not only make strong hydrophobic interactions with inhibitor, but also act as gatekeeper to regulate the opening of channel. The present study provides important insights into the structure-function relationships of three cytochrome P450s and the molecular basis for development of potent inhibitors.

Sustained release timolol maleate loaded ocusert based on biopolymer composite.

In the present investigation, the effect of timolol maleate loaded ocuserts was studied as an alternative for conventional anti-glaucoma formulation. Ocuserts were prepared using natural polymer sodium alginate and ethyl cellulose. Physico-chemical properties along with drug entrapment efficiency (94-98%), content uniformity (93.1% ±â€¯0.264-98.00% ±â€¯0.321), in vitro drug release (83.42% ±â€¯0.35 at end of 12 h), ex vivo permeation all showed satisfactory results, which was found to follow zero order kinetics. Ex vivo permeation studies showed better results, revealed that the permeability coefficient was dependent on polymer type. The sterility test accelerated stability studies and in vivo studies such as eye irritancy test, in vivo drug release of the optimized ocusert was determined. The anti-glaucoma activity was measured using Schiotz tonometer at different time interval. Significant reduction in Intra ocular pressure (IOP) within 3 days was observed in case of rabbits treated with ocusert in comparison to the rabbit treated with marketed eye drop formulation. Hence timolol maleate loaded ocuserts proved to be a promising and viable alternative over conventional eye formulation for the sustained and controlled ophthalmic drug delivery, targeting the drug within the ocular globe thus improving patient compliance for the treatment of glaucoma.

Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.

Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K+ channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.

Synthesis and evaluation of mucoadhesive acryloyl-quaternized PDMAEMA nanogels for ocular drug delivery.

Poly((2-dimethylamino)ethyl methacrylate) (PDMAEMA) nanogels were synthesized via surfactant-free free-radical polymerization technique in aqueous conditions utilizing N,N'-methylene-bis-acrylamide (MBA) as a crosslinking agent. The PDMAEMA nanogels were subsequently quaternized with acryloyl chloride in order to yield mucoadhesive materials which incorporate two mucoadhesive concepts; electrostatic interactions and covalent bond forming acrylate groups. The native PDMAEMA nanogels were found to exhibit good mucoadhesive properties on ex vivo bovine conjunctival tissues, which was found to increase proportionally with the degree of quaternization. With a view to determine the ocular drug delivery capabilities of the materials, both quaternized and native nanogels were loaded with pilocarpine hydrochloride via an absorption method, and their in vitro release profiles were analysed. The nanogels were found to exhibit a high loading capacity (>20% of total weight) and a sustained release over 6h.

Effect of gellan gum on the thermogelation property and drug release profile of Poloxamer 407 based ophthalmic formulation.

The effect of gellan gum on the gelation behavior and in-vitro release of a specific drug named pilocarpine hydrochloride from different ophthalmic formulations based on poloxamer 407 is examined. The mixture of 0.3wt% gellan gum and 18wt% poloxamer (PM) solutions show a considerable increase in gel strength in physiological condition. Gel dissolution rate from PM based formulation is significantly decreased due to the addition of gellan gum. FTIR spectra analysis witnesses an interaction in between OH groups of two polymers which accounts for lowering in gelation temperature of PM-gellan gum based formulations. It is also observed from the cryo-SEM study that the pore size of PM gel decreases with an addition of gellan gum and in-vitro release studies indicate that PM-gellan gum based formulation retain drug better than the PM solution alone. Therefore, the developed formulation has the potential to be utilized as an in-situ ophthalmic drug carrier.

Longer latency of sensory response to intravenous odor injection predicts olfactory neural disorder.

A near loss of smell may result from conductive and/or neural olfactory disorders. However, an olfactory test to selectively detect neural disorders has not been established. We investigated whether onset latency of sensory response to intravenous odor injection can detect neural disorders in humans and mice. We showed that longer preoperative onset latency of odor recognition to intravenous odor in patients with chronic rhinosinusitis predicted worse recovery of olfactory symptoms following sinus surgery. The onset latency of the olfactory sensory neuron (OSN) response to intravenous odor using synaptopHluorin signals from OSN axon terminals was delayed in mice with reduced numbers of OSNs (neural disorder) but not with increased mucus or blocked orthonasal pathways (conductive disorders). Moreover, the increase in onset latency correlated with the decrease in mature OSN numbers. Longer onset latency to intravenous odor injection is a useful biomarker for presence and severity of olfactory disorders with neural etiology.

A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor.

G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.

General Pharmacokinetic Model for Topically Administered Ocular Drug Dosage Forms.

PURPOSE: In ocular drug development, an early estimate of drug behavior before any in vivo experiments is important. The pharmacokinetics (PK) and bioavailability depend not only on active compound and excipients but also on physicochemical properties of the ocular drug formulation. We propose to utilize PK modelling to predict how drug and formulational properties affect drug bioavailability and pharmacokinetics. METHODS: A physiologically relevant PK model based on the rabbit eye was built to simulate the effect of formulation and physicochemical properties on PK of pilocarpine solutions and fluorometholone suspensions. The model consists of four compartments: solid and dissolved drug in tear fluid, drug in corneal epithelium and aqueous humor. Parameter values and in vivo PK data in rabbits were taken from published literature. RESULTS: The model predicted the pilocarpine and fluorometholone concentrations in the corneal epithelium and aqueous humor with a reasonable accuracy for many different formulations. The model includes a graphical user interface that enables the user to modify parameters easily and thus simulate various formulations. CONCLUSIONS: The model is suitable for the development of ophthalmic formulations and the planning of bioequivalence studies.