Forskolin: upcoming antiglaucoma molecule.

Forskolin is the first pharmaceutical drug and product derived from a plant to be approved in India by the DCGI in 2006. Forskolin (7beta-acetoxy-8, 13-epoxy-1a, 6ß, 9a-trihydroxy-labd-14-en-11-one) is a diterpenoid isolated from plant Coleus forskohlii (Lamiaceae). It is a lipid-soluble compound that can penetrate cell membranes and stimulates the enzyme adenylate cyclase which, in turn, stimulates ciliary epithelium to activate cyclic adenosine monophosphate, which decreases intraocular pressure (IOP) by reducing aqueous humor inflow. The topical application of forskolin is capable of reducing IOP in rabbits, monkeys, and humans. In its drug interactions, forskolin may act synergistically with epinephrine, ephedrine and pseudoephedrine. Whereas the effects of anti-clotting medications like warfarin, clopidogre, aspirin, anoxaparin, etc., may be enhanced by forskolin. Forskolin is contraindicated in the medications for people with ulcers as forskolin may increase acid level. Forskolin has a very good shelf-life of five years. Recently, its Ophthalmic inserts and in situ gels for sustained and delayed-release drug delivery systems were tested in New Zealand Albino Rabbits for its antiglaucoma efficacy. This drug review explains Forskolin as a drug, its antiglaucoma potential and recent findings of forskolin as an antiglaucoma agent. The literature search method used for this review was different databases and search engines like PubMed, International Pharmaceutical Abstracts, Google, Medicinal and Aromatic Plants (MAPA).

The classical competitive antagonism of phentolamine on smooth muscle preparations, investigated by two procedures.

1. In isolated smooth muscle tissues taken from rats, rabbits and guinea-pigs, all at 37.5 degrees C, the equilibrium dissociation constant (K(beta)) of the competitive, reversible alpha-adrenoceptor antagonist phentolamine varied between 4 and 28 nm. 2. The concentration of the antagonist required to inhibit contractions to direct- or indirect-acting alpha-adrenenoceptor agonists by 50% (IC50) also varied between 5 and 30 nm. 3. From one tissue to another, the IC50/K(beta) ratio of the blocker varied from 1 to 2.5, the values being close to those predicted by classical receptor theory based on the law of mass action. 4. At 27.5 degrees C, using phenylephrine as the spasmogen in rat aorta, the IC50/K(beta) ratio for phentolamine was 3.1. 5. A significantly higher IC50 compared with K(beta) for phentolamine indicates that the procedures for estimating affinity constants for a competitive antagonist are not equivalent.

Antagonism by imidazoline-type drugs of muscarinic and other receptors in the guinea-pig ileum.

1 Several imidazolines were examined for the antagonism of muscarinic (M3) and other receptors on the isolated ileum of guinea-pig. The effect of the muscarinic agonist, carbachol was competitively antagonized by oxymetazoline at 10(-5) m. A dissociation constant (KB) of 3.6 microm for the antagonist was calculated. At higher concentrations, 3 x 10(-5) and 10(-4) m, of the antagonist, the agonist dose-response curve was shifted to the right with a decrease in the maximum effect. Thus, a non-competitive block occurred at higher concentrations of oxymetazoline. Blockade of histamine H, and serotonin receptor-mediated responses by oxymetazoline were also of a non-competitive type. 2 Naphazoline at 10(-4) m shifted the dose-response curves of carbachol and serotonin to the right by two- and 15-fold, respectively. The maximum contraction of the agonist was not affected. Tolazoline also had a weak antihistaminic activity. At similar concentration; tetrahydrozoline clonidine and phentolamine at 10(-5) m produced two-, three- and four-fold shift of the carbachol dose-response curve without significant changes in the maxima. Neither methoxamine, p-amino-clonidine nor cimetidine blocked the responses of carbachol. 3 The isosteric nature of the alpha-adrenoceptor agonist, oxymetazoline and some imidazolines with carbachol, in part, explains its molecular competition at the muscarinic M3 receptor of the guinea-pig ileum. Surprisingly, contractile effects of carbachol (M3), histamine (H1) or serotonin (5HT3/5HT4) were not influenced by methoxamine, tetrahydrozoline, p-amino clonidine and cimetidine.

Biotransformations with Rhizopus arrhizus and Geotrichum candidum for the preparation of (S)-atenolol and (S)-propranolol.

(+/-)-Atenolol/(+/-)-propranolol and their acetates were incubated with the fungus Rhizopus arrhizus and Geotrichum candidum separately for different time intervals to afford (S)-atenolol/(S)-propranolol in good optical yield. The time and pH for this biotransformation was optimised. The present biodegradations using Rhizopus arrhizus and Geotrichum candidum provides a simple and useful method to obtain (S)-atenolol and (S)-propranolol which are active enantiomers of the beta-adrenergic blockers.

Depolarization of membrane potential and the smooth muscle contraction by isothiocyanatobenzyl imidazoline in guinea-pig stomach circular muscle.

Isothiocyanatobenzyl imidazoline (IBI) produces characteristic slowly developing contraction of many smooth muscle preparations including the circular smooth muscle of the guinea-pig stomach. Changes in the membrane potential were recorded intracellularly, and the muscle contraction induced by IBI was investigated. IBI at 100 micromol/l slowly produced a sustained depolarization of the membrane with a maximum change of approximately 15 mV. This depolarization could not be blocked by 1-hyoscyamine, 100 nmol/l. An imidazoline analogue, oxymetazoline at 1 micromol/l, did not change the resting membrane potential as observed after IBI. Significant membrane depolarization after IBI still occurred in Ca2+-free medium. During IBI-induced depolarization, sudden reduction of Na+ to 30 mmol/l in the medium reduced the depolarization slightly. IBI-induced depolarization was additive with that produced by 20 mmol/l K+ in the medium. In the presence of tetraethylammonium chloride or levcromakalim or nifedipine, IBI continued to depolarize the membrane although functional pharmacological experiments showed that the contractile effects of IBI were significantly inhibited by 30 micromol/l levcromakalim and abolished by 100 nmol/l nifedipine. At 100 micromol/l phentolamine (reported by others as an inhibitor of ATP-sensitive potassium channels) completely blocked IBI-induced contraction. Phentolamine (30 micromol/l) blocked the contractile effects of IBI by 50%. On the other hand, S(-)-Bay K 8644, a voltage-dependent calcium channel activator, was additive with the contractile response of IBI. These results indicated that IBI produced membrane depolarization and contraction of the guinea-pig stomach circular muscle, by a mechanism not involving muscarinic receptors or alpha-adrenoceptors. Even though levcromakalim, an ATP-sensitive potassium channel opener, could not inhibit IBI-induced depolarization, the ATP-sensitive potassium channel and the voltage-dependent calcium channel may be intrinsically linked with the action of IBI.

Enhanced sensitivity of the iris sphincter to the muscarinic agonist carbachol at lower temperature.

Cooling of the human iris sphincter from 37.5 degrees C to 17.5 degrees C potentiates the concentration response curves of the muscarinic agonist, carbachol. Although EC50 value 0.31 micromol/l of the agonist was not significantly affected at the lower temperature, the maximum contraction of the control at 37.5 C was potentiated by 263%. Such potentiation was 272%, 135% and 125% for the dog, horse and pig in sphincter, respectively. Low temperature potentiation of the carbachol can be reversed by warming the tissue to 37.5 degrees C. Pretreatment of the tissue with competitive muscarinic receptor blocker, atropine, blocked the contractile action of carbachol at 37.5 degrees C and 17.5 degrees C which resulted in nearly equal KB of approximately 2 nmol/l, but at 17.5 degrees C, the contraction induced by carbachol was antagonized by atropine with difficulty to give pseudo KB of 67 nmol/l. Ideally, a competitive reversible antagonist is expected to give equal KB values for the drug receptor interaction; irrespective of the method of study. The higher KB value indicates a functional antagonism of the agonist activated cascade by the blocker. Clinical implications of the drug antagonism at low temperatures are discussed.

Selectivity of muscarinic agonists including (+/-)-aceclidine and antimuscarinics on the human intraocular muscles.

The average EC50 value and the maximum response of carbachol on the human circular ciliary muscle obtained within 24 h of postmortem hypoxia was 517 nmol/l and 135 mg, respectively. These values for carbachol did not differ significantly from that of the longitudinal ciliary muscle. However, when tested at 1 mumol/l of carbachol, the peak response of the longitudinal muscle occurred at 59 sec vs 173 sec for that of the circular muscle of 70 year old donors. The relative potency of the muscarinic agonists on the circular muscle was oxotremorine-M, 1 > carbachol, 1/4 > pilocarpine, 1/19 > aceclidine, 1/132. The relative order of potency of agonists was similar for the longitudinal muscle. Only pilocarpine and aceclidine were partial agonists which produced 80-85% of the maximum response. When compared with the EC50 values of aceclidine on the iris sphincter and the longitudinal ciliary muscles, the agonist potency was only 1/28 for the latter tissue. Implications of these findings in relation to the use of these agonists in glaucoma are discussed. The pKB values of muscarinic antagonists on the circular ciliary muscle were: atropine, 8.8; cyclopentolate, 7.8; tropicamide, 7.4; P.F. HHSiD, 7.0; pirenzepine, 6.4; and methoctramine, 5.7. Nearly equal pKB values of each antagonist were obtained for the longitudinal ciliary muscle and iris sphincter. Based on the affinity constants of various competitive antagonists, the human iris as well as ciliary muscles may contain M3, M2 or M4 subtypes of muscarinic receptors.

Isothiocyanatobenzyl imidazoline is an alkylating agent for I2-imidazoline binding sites in rat and rabbit tissues.

Isothiocyanatobenzyl imidazoline (IBI), the 4'-NCS analogue of tolazoline, has been used to alkylate several receptor sites in rabbit iris muscles. Because of the high affinity of tolazoline for the I2-imidazoline binding sites (Ki = 16-130 nM), this study was designed to assess whether IBI is also an alkylating agent for these sites. In competition studies, IBI displayed moderate affinity (Ki approximately 2-3 microM) against I2A-imidazoline sites in the rabbit cerebral cortex and I2B-imidazoline sites in the rat cerebral cortex labelled by [3H]2-(2-benzofuranyl)-2-imidazoline ([3H]2-BFI). However, preincubation (30 min at 25 degrees C) of rat cortical and liver membranes with IBI (10(-7) M to 10(-3) M), followed by extensive washing, markedly decreased (17% to 96%) the specific binding of [3H]2-BFI to I2B-imidazoline sites. IBI (10(-5) M to 10(-3) M) also bound irreversibly to I2A-imidazoline sites in rabbit cerebral cortex but with a lesser efficacy (27% to 83% reduction of [3H]2-BFI binding). Saturation curves of [3H]2-BFI binding in the rat cerebral cortex indicated that preincubation with 10(-6) M IBI reduced the total density (Bmax) without affecting the affinity (Kd) of I2B-imidazoline sites for IBI. Acute treatments (6 h) with IBI (10 and 30 mg/kg, i.p.) also dose-dependently reduced (26% and 41%; respectively) the total density of I2B-imidazoline sites. These results demonstrate the ability of IBI to alkylate I2-imidazoline binding sites in vitro and in vivo and provide evidence for the use of IBI as a new tool for the study of the functional implications of imidazoline binding sites.

Medetomidine analogs as alpha 2-adrenergic ligands. 3. Synthesis and biological evaluation of a new series of medetomidine analogs and their potential binding interactions with alpha 2-adrenoceptors involving a "methyl pocket".

The synthesis and the biological evaluation of a new series of medetomidine analogs are reported. The substitution pattern at the phenyl ring of the tetralin analogs had a distinct influence on the alpha 2-adrenoceptor binding affinity. 4-Methylindan analog 6 was the most potent alpha 2-adrenoceptor binding ligand among these 4-substituted imidazoles, and its alpha 2-adrenoceptor selectivity was greater than the 5-methyl tetralin analog 4c. Ligand-pharmacophore and receptor modeling were combined to rationalize alpha 2-adrenoceptor binding data of the imidazole analogs in terms of ligand-receptor interactions. The structure-activity relationships that were apparent from this and previous studies were qualitatively rationalized by the binding site models of the alpha 2-adrenoceptor. The benzylic methyl group of medetomidine or the naphthyl analog 2a was superimposable with the alpha-methyl group of (-)-alpha-methylnorepinephrine and fit into the proposed "methyl pocket" of the alpha 2-adrenoceptor defined by the residues Leu110, Leu169, Phe391, and Thr395.

Comparison of post-junctional alpha-adrenoceptors in iris dilator muscle of humans, and albino and pigmented rabbits.

The relative potency of alpha-adrenoceptor agonists and the dissociation constants of competitive antagonists were studied to characterize the post-junctional alpha-adrenoceptor of the human iris dilator muscle. The data obtained from human iris dilator tissue was compared to that from rabbit. The iris dilator muscle was mounted in an organ bath and tension changes were recorded. (-)-Norepinephrine, (-)-phenylephrine (PE), oxymetazoline and p-aminoclonidine caused contractile responses in albino rabbit, pigmented rabbit and human iris dilator muscle in a concentration-dependent manner. The imidazoline molecules were partial agonists. In rabbit iris dilator, desensitization occurred to repeated oxymetazoline application at an interval of 1 h but recovery to the agonist activity was complete in about 3 h. Exposure to cocaine (10 mumol/l), hydrocortisone (100 mumol/l) and U-0521, a catechol-O-methyltransferase inhibitor (100 mumol/l), significantly potentiated the response to norepinephrine by 92-, 32- and 7 fold in iris dilator tissue of albino rabbit, pigmented rabbit and human, respectively. After block of "uptake1" and "uptake2", the EC50 values of norepinephrine in the albino rabbit, pigmented rabbit and human iris dilator did not differ and ranged from 99 to 195 nmol/l. Small but significant potentiation by uptake blockers was also observed in the responses to PE in the albino rabbit or pigmented rabbit iris dilator. The average maximum tension induced by 100 mumol/l PE was 96 +/- 11 mg (n = 10), 197 +/- 11 mg (n = 11), 45 +/- 5 mg (n = 27) in albino rabbit, pigmented rabbit and human iris dilator, respectively. In human iris dilator, the responses to PE were competitively antagonized by prazosin, 5-methylurapidil and phentolamine with apparent pKB values of 7.3, 6.6 and 7.5, respectively. The pKB values of the prazosin-PE interaction in iris dilator of albino and pigmented rabbit were 8.6 and 6.4, respectively. These results suggest that the post-junctional alpha-adrenoceptors in iris dilator may be similar to that in pigmented rabbit iris. The alpha-adrenoceptor of the human or pigmented rabbit iris dilator may be characterized as alpha 1L-adrenoceptor subtype. The alpha-adrenoceptor of albino rabbit iris dilator appears to be a high affinity subtype. Furthermore, albino rabbit may not be the best strain for the drug research which is relevant to human ocular therapeutics.

Irreversible agonist and antagonist properties of isothiocyanatobenzyl imidazoline in albino rabbit iris muscles.

The alkylating agent isothiocyanatobenzyl imidazoline (IBI) was synthesized to investigate the unique receptor interacting properties of imidazolines. On the isolated rabbit iris sphincter, IBI produced concentration-dependent responses with an EC50 of 18 mumol/1, and at the highest concentration tested the maximum contraction of the tissue was 50% of the carbachol maximum. At equiactive concentrations with the similar washing procedure, the total duration of responses to IBI and carbachol was 24 and 3 min, respectively. After repeated washing, the sphineter relaxes to the control baseline of tone but, after reexposure to IBI for 6 h, failed to contract, indicating that desensitization or irreversible block has developed. Unlike with carbachol, the sphincter contraction to IBI was not affected by atropine 1 mumol/1, indomethacin 1 mumol/1, verapamil 10 mumol/1, or nifedipine 10 mumol/1. At a higher concentration of nifedipine and papaverine 100 mumol/1, the response to IBI was blocked. Furthermore, the contractile response to IBI was abolished by Ca++ removal from the medium. Under similar conditions, 26 +/- 8% of the maximum response to carbachol was preserved. Thus influx of extracellular as well as rise in intracellular Ca++ appears vital for the contractile response to IBI. IBI did not contract the iris dilator, but shifted the concentration-response curve to the alpha-adrenoceptor activator, phenylephrine, to the right with a reduction in the maximum response. The tissue failed to regain the sensitivity to phenylephrine after 6 h of repeated washing. Phentolamine and nifedipine provided a small but significant protection of the response to phenylephrine against the irreversible block by IBI. Based on chemical and pharmacological properties of IBI, it is concluded that the molecule acts in the rabbit as an irreversible agonist on unidentified receptors of the iris sphineter and an irreversible antagonist of multiple receptors on the iris dilator. These molecular properties of IBI are clearly different from that of the parent imidazoline molecule tolazoline.

Pharmacologic quantitation.

Either in vivo or in vitro quantitatives comparisons of drugs based on the dose-response relations were known for a long time. For therapeutically targeted molecular structure activity studies, agonists are simply compared on the basis of concentrations eliciting half-maximum response (EC50) and per cent maximum response elicited by drugs in the given system. For partial agonists the receptor affinity determined as the dissociation constant (KA) in homogenates corresponds to that in the organ system. However, due to the presence of spare receptors the EC50 of potent agonists does not represent the KA. If a fraction of receptors are irreversibly inactivated in the tissue, KA as well as relative intrinsic efficacy of the potent agonists can be obtained. Although quantitation of irreversible antagonists is quite complex, the competitive reversible blockers can be accurately compared on the basis of equilibrium dissociation constant (KB) values. Along with the relative order of potency of agonists and KB values of antagonists, receptors can be characterized and subclassified. Preclinical Therapeutic Index of drugs and Toxicity Index of pesticides require determinations of mean lethal dose (LD50) and the mean effective dose (ED50) in laboratory animals, so that a relatively safe drug can be promoted for human use. The understanding of pharmacokinetics is essential to explain drug action in the target organs. Drug combinations are investigated by isobolorographic analysis.

Medetomidine analogs as alpha 2-adrenergic ligands. 2. Design, synthesis, and biological activity of conformationally restricted naphthalene derivatives of medetomidine.

A new series of naphthalene analogs of medetomidine have been prepared and evaluated for their alpha-adrenergic activities. The methylnaphthyl analog 5a showed significant selectivity for alpha 2-adrenoceptors and behaved as a partial alpha 1-agonist in rat aorta preparations. In contrast, the Z-ethylene analog 8c was alpha 1-selective and behaved as a potent alpha 1-antagonist. Two rigid analogs (6 and 7) exhibited large differences in binding affinities at alpha 1-VS alpha 2-receptors, indicating that the conformational flexibility of 5a is important for the fulfillment of the alpha-adrenergic activities. Molecular modeling studies began with conformational analysis of classical phenethylamines and medetomidine analogs. Superimposition of medetomidine conformations with those of phenethylamines provided a tentative explanation for the alpha 2-adrenergic activity of the new imidazoles. A common binding mode for phenethylamines and imidazoles with alpha 2-adrenoceptors is proposed. Knowledge of the biological properties of the 4-substituted imidazoles, integrated with the information derived from computer-assisted molecular modeling, has provided new insights for the structural and conformational requirements of this class as new adrenergic drugs.

Stereoselective modification of circular dichroism spectra of rat lung beta-adrenoceptor protein preparation by enantiomers of epinephrine.

The presence of beta-adrenoceptor in a rat lung membrane preparation was confirmed by stereoselective competition of enantiomers of epinephrine with labeled iodocyanopindolol. The receptor-rich protein fraction, when combined with the pharmacologically active (-)-epinephrine, exhibited specific changes in the 205-220 nm region of circular dichroism spectra, indicating that the receptor helices may be perturbed. The (+)-epinephrine combined with the lung protein produced little or no change of the spectra. Bovine serum albumin, when combined with either enantiomer of epinephrine, produced nonstereoselective alterations of the spectra. Thus, the data provide important evidence for the higher intrinsic pharmacologic activity of the natural (-)-epinephrine over the unnatural (+)-enantiomer.

Actions of imidazolines on bovine ciliary artery.

A segment of bovine ciliary artery exhibits nonlinear biphasic contractile responses to agonists. The maximal responses of oxymetazoline (OMZ) and norepinephrine (NE) were 55% and 45%, respectively (KCl = 100%). The relative potency comparison based on the ED50 of the first component of the dose-response curve was OMZ; 1 > NE; 1/7 > phenylephrine; 1/72. At equipotent doses of NE and OMZ, the t1/2 duration of response of the latter agonist was 43 times greater than that of the former and, after 3 hrs, responses were 95% reproducible. Naphazoline, tetrahydrozoline and para-aminoclonidine did not produce significant contraction of the blood vessel but did antagonize the action of the agonists. As compared to that of NE, naphazoline was selective in blocking responses of OMZ. Phentolamine blocking action of NE was greater than that of OMZ. Nifedipine, 100 nM, reduced the second component of OMZ response. Although the vascular action of NE was blocked by prazosin, the contractile action of OMZ was totally resistant to block by 1 microM of prazosin. These results indicate that ciliary artery contains a subtype of alpha-adrenoceptors (alpha 11) with low sensitivity to prazosin. The tissue also contains a unique OMZ sensitive site or imidazoline receptors which exhibit low affinity to alpha-adrenoceptor blockers.

Infectivity of algal viruses studied by chlorophyll fluorescence.

Algal virus infection proceeds via the specific recognition of the host cell wall, penetration of the cell wall and transfer of genetic material into the cytoplasm of the host cell. This process is similar to that which occurs when bacteriophage infect bacteria so that techniques and concepts developed to study bacteriophage are applicable to algal virus studies. By measuring virus-induced changes in chlorophyll fluorescence we have redefined classical studies on the distribution of infectivity. We show that infectivity does not follow a Poisson distribution with a fixed mean, n. By analysing the infectivity of algal viruses over a broad range of virus:cell ratios we have obtained a corrected Poisson distribution that reflects the probability of multiple virus particles attached per cell and is equally applicable to algal viruses and bacteriophage.

Synthesis and biological activities of a new set of irreversibly acting 2-(4'-isothiocyanatobenzyl)imidazoline analogs in rat thoracic aorta.

IBI [2-(4'-isothiocyanatobenzyl)imidazoline, 3] has been shown to cause slow-onset, long-lasting contractions of rat thoracic aorta through a non-alpha-adrenergic receptor (non-alpha-AR) mediated mechanism. A series of IBI-related anlogs 7-14 and 16 was prepared to determine the structural requirements for the interaction with non-alpha-AR in rat aortic strips. All IBI analogs produced concentration-dependent contractile responses on rat thoracic aorta. Whereas the actions of analogs 7, 14, and 16 were partly mediated by alpha-ARs, the stimulatory activities of the remaining IBI analogs were unaffected by phenoxybenzamine pretreatment, suggesting that a non-alpha-adrenergic mechanism is involved. We have shown that the contractile actions of IBI and analogs 10-13 were not blocked with the imidazoline/guanidinium receptive site (IGRS) ligands idazoxan, cirazoline, or clonidine. However, the calcium channel blockers nifedipine or verapamil shifted the concentration-response curve of IBI and its analogs 10-13 to the right and reduced the maximal contractile responses. The action of IBI on rat thoracic aorta was reduced by the omission of extracellular calcium in the medium. These results suggest that the stimulatory activities of IBI and analogs 10-13 are not related to the activation of alpha-AR or IGRS receptors and are likely coupled to the voltage-dependent Ca2+ channels.

Relevance of drug-melanin interactions to ocular pharmacology and toxicology.

In melanocytes, the biosynthesis of L-dopa derived indole polymer, melanin, is accelerated by tyrosinase and related enzymes. The brown to black pigment is characterized by a stable free-radical property. In humans, a pigment dependent slow onset of ocular actions of ephedrine, atropine, cocaine, pilocarpine and related medications was observed. Extensive accumulation of drugs by melanin appears to be the most important factor governing the long term therapeutic/toxicological activities. Drugs crossing placental circulation are localized in the mouse fetal eye. Thus, drugs exhibit a high binding capacity for melanin containing tissues. Studies on synthetic melanin and melanin granules also indicated a high binding capacity of many therapeutic classes of drugs, including psychotropics. In addition to the liposoluble property of the molecule, there is a definite relationship between chemical structure and the affinity of drugs for melanin. For example, the affinity of chlorpromazine for melanin is higher than that of chlorprothixene. NMR studies, with soluble melanins indicate that there is a steric preference among ephedrine enantiomers. A high binding capacity indicates that more than two molecules of (-)-ephedrine may complex with one indole unit of melanin. Ocular drug development calls for the study of qualitative and quantitative aspects of drug-melanin interaction.