An electrophoretic method to deliver topical drugs to the eye

It is possible that many patients avoid complete ophthalmic exams because pupil dilation is slow, reversal sluggish, and vision blurred. Others experience incomplete dilation during exams or prior to surgery when good dilation is essential to successful outcome. Iontophoresis, the application of low-level electrical current to promote traversal of desired molecules across a boundary, has been used for many years and has recently become common in transdermal drug delivery. We now investigate iontophoresis as a method of accelerating drug absorption into the ocular anterior segment. In vivo rabbit studies assessed iontophoresis effects on the performance of dilators and constrictors. 1-mA and 4-mA direct current levels applied for 2-minute durations yielded dilation time-history measurements. Subsequent in vitro tests at a wide range of current densities showed minimal chemical modifications in ocular pharmaceuticals. Drug samples processed through high-performance liquid chromatography (HPLC) pinpointed minimal structural changes. Detailed in vivo rabbit testing is under way. Using 2 dilators and constrictors in crossed testing with 0.5-mA to 1.25-mA current levels and 20-sec to 60-sec durations, we recorded dilation progress by digital photography. Initial studies showed faster, larger dilations and quicker reversal using iontophoresis. Drug testing showed chemical structures remaining constant for clinically useful current levels, < or = 1 mA (< or = 1.25 mA/cm2 current density). Drug pH and HPLC retention times were constant within this range, and resistivity varied linearly as expected for increasing current. Rabbit testing will quantify improved drug speed and efficacy, validate the charge delivery electrode design, and indicate iontophoretic current and duration for further use. Tested ocular drugs showed no degradation when exposed to clinically useful iontophoretic currents. Preliminary results indicate significant time reductions for dilation and reversal, plus increases in maximum dilation. This procedure may aid clinicians by allowing more rapid complete examinations and surgical preparations for patients. Making dilation more convenient will also improve patient acceptance of exams, aiding earlier detection and treatment of ocular disease.     

Exocytosis mechanism as a new targeting site for mechanisms of action of antiepileptic drugs

Carbamazepine (CBZ) and zonisamide (ZNS) are effective antiepileptic drugs (AEDs) for the treatment of epilepsy and mood disorder. One of the mechanisms of action of CBZ and ZNS is inactivation of voltage-gated Na+ channel (VGSC). However, the major mechanism(s) of action of these AEDs is not clear yet. We have been exploring novel targeting mechanisms for the antiepileptic actions of CBZ and ZNS during the past ten years. In this report, we describe our hypothesis regarding the new targeting mechanisms for the antiepileptic action of AEDs. We determined an interaction between these AEDs and inhibitors of both voltage-sensitive Ca2+ channels (VSCCs) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) on neurotransmitter exocytosis using microdialysis. Perfusion with therapeutic concentrations of CBZ and ZNS increased basal neurotransmitter release. This stimulatory action was predominantly inhibited by inhibitors of N-type VSCC and syntaxin. CBZ and ZNS increased Ca2+-evoked release, an action selectively inhibited by inhibitors of N-type VSCC and syntaxin. CBZ and ZNS reduced K+-evoked release, an action predominantly inhibited by inhibitors of P-type VSCCs and synaptobrevin. These actions of CBZ and ZNS on neurotransmitter exocytosis could be observed under the condition of inhibition of VGSC using perfusion with tetrodotoxin. Our findings enhance our understanding of the mechanisms of action of CBZ and ZNS as AEDs, which possibly reduce P-type VSCCs/synaptobrevin-related exocytosis mechanisms during the depolarization stage, and simultaneously enhance N-type VSCCs/syntaxin-related exocytosis mechanisms at the resting stage.     

General patterns and asymptotic dose in the design of coated stents

Stents are used in interventional cardiology to keep a diseased vessel open. New stents are coated with a medicinal agent to prevent early reclosure due to the proliferation of smooth muscle cells. It is recognised that it is the dose of the agent that effectively controls the growth. This paper focusses on the asymptotic behaviour of the dose for general families of coated stents under a fixed ratio between the coated region of the stent and the targeted region of the vessel and set therapeutic bounds on the dose. It generalises the results of Delfour, Garon and Longo for stents made of a sequence of thin equally spaced rings to stents with an arbitrary pattern. It gives the equation of the asymptotic dose for a normal tiling of the target region using the theory of tilings, patterns and motifs on a cylinder.     

Transport of ABA from the site of biosynthesis to the site of action

There is substantial evidence that abscisic acid (ABA) moves within plants. ABA has been considered as a root-derived signaling molecule that induces stomatal closure in response to dry soil conditions. It has been also reported that ABA synthesized in vegetative tissues is translocated to the seeds. The transport of ABA is an important factor in determining the endogenous concentrations of the hormone at the site of action, and hence, it is an important process in physiological responses. However, the molecular mechanisms that regulate ABA transport are not fully understood. Recent studies using Arabidopsis indicate that ABA is actively synthesized in leaf vascular tissues in response to drought, and that ABA is subsequently transported to the guard cells to close stomata. Identification of the transporters that mediate ABA export from the inside to the outside of the cells at the site of ABA biosynthesis (vascular tissues) and ABA uptake into the cells at the site of action (guard cells), respectively, in this species indicates an active mechanism to regulate ABA transport. Although Arabidopsis represents only one model plant, these findings are useful to discuss common or different regulatory mechanisms among different species and to improve our total understanding of the regulation of ABA transport.     

Galanin: evidence for a hypothalamic site of action to release growth hormone

Galanin is a 29 amino acid peptide that was isolated and characterized from porcine intestinal extracts. The presence of galanin-like immunoreactivity in neuronal elements in the hypothalamus and median eminence suggested a role for it in the hypothalamic control of anterior pituitary function. A hypothalamic site of action of galanin to stimulate growth hormone (GH) release is suggested by our observation that doses as low as 50 picomoles when infused into the third cerebroventricle of conscious, unrestrained ovariectomized rats resulted in significantly elevated plasma levels of GH. This effect was specific for GH and was dose-related. The failure of galanin to alter GH release from dispersed, cultured anterior pituitary cells in vitro further suggests that endogenous galanin plays a neuromodulatory role at the level of the median eminence, possibly affecting the release of known GH-releasing and/or inhibiting factors.     

Bilayer interactions of pHLIP, a peptide that can deliver drugs and target tumors

The pH-dependent insertion of pHLIP across membranes is proving to be a useful property for targeting acidic tissues or tumors and delivering drugs attached to its C-terminus. It also serves as a model peptide for studies of protein insertion into membranes, so further elucidation of the insertion mechanism of pHLIP and its features is desirable. We examine how the peptide perturbs a model phosphatidylcholine membrane and how it associates with the lipid bilayer using an array of fluorescence techniques, including fluorescence anisotropy measurements of TMA-DPH anchored in bilayers, quenching of pHLIP fluorescence by brominated lipids and acrylamide, and measurements of energy transfer between aromatic residues of pHLIP and TMA-DPH. When pHLIP is bound to the surface of bilayers near neutral pH, the membrane integrity is preserved whereas the elastic properties of bilayers are changed as reported by an increase of membrane viscosity. When it is inserted, there is little perturbation of the lipids. The results also suggest that pHLIP can bind to the membrane surface in a shallow or a deep mode depending on the phase state of the lipids. Using parallax analysis, the change of the penetration depth of pHLIP was estimated to be 0.4 Å from the bilayer center and 2.8 Å from the membrane surface after the liquid-to-gel phase transition.     

Opioids and central baroreflex control: a site of action in the nucleus tractus solitarius

These studies investigated whether the nucleus of the tractus solitarius (NTS) is a central site where opioids modulate baroreceptor reflexes. Microinjections into the NTS of [D-Ala2,MePhe4, Gly-ol5]enkephalin (DAGO) significantly reduced reflex-mediated depressor responses evoked by electrical stimulation of the aortic nerve. Subsequent NTS injections of naloxone restored baroreflexes to control levels. These results demonstrate that the NTS is a central site where exogenously administered opioids can modulate baroreceptor reflexes. NTS injections of naloxone had no effect on baroreflex function, suggesting that tonic activation of opioid receptors at this site plays little or no role in central baroreflex control.     

Improving smooth muscle cell exposure to drugs from drug-eluting stents at early time points: a variable compression approach

The emergence of drug-eluting stents (DES) as a viable replacement for bare metal stenting has led to a significant decrease in the incidence of clinical restenosis. This is due to the transport of anti-restenotic drugs from within the polymer coating of a DES into the artery wall which arrests the cell cycle before restenosis can occur. The efficacy of DES is still under close scrutiny in the medical field as many issues regarding the effectiveness of DES drug transport in vivo still exist. One such issue, that has received less attention, is the limiting effect that stent strut compression has on the transport of drug species in the artery wall. Once the artery wall is compressed, the stents ability to transfer drug species into the arterial wall can be reduced. This leads to a reduction in the spatial therapeutic transfer of drug species to binding sites within the arterial wall. This paper investigates the concept of idealised variable compression as a means of demonstrating how such a stent design approach could improve the spatial delivery of drug species in the arterial wall. The study focused on assessing how the trends in concentration levels changed as a result of artery wall compression. Five idealised stent designs were created with a combination of thick struts that provide the necessary compression to restore luminal patency and thin uncompressive struts that improve the transport of drugs therein. By conducting numerical simulations of diffusive mass transport, this study found that the use of uncompressive struts results in a more uniform spatial distribution of drug species in the arterial wall.     

Redesigning the active site of a carboxyl esterase from the archaeon Archaeoglobus fulgidus to improve sensitivity to organophosphorus compounds

Organophosphorus compounds (OPs) are widely used as pesticides because of their ability to inhibit the activity of acetylcholinesterase (AChE) in the nervous system. Thus, AChE is generally used as a biosensor for pesticide detection. Due to the instability of AChE a more stable enzyme would be desirable for robust applications. We investigated the sensitivity of a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus (AFEST) to seven selected OPs. The IC₅₀ of dichlorvos against AFEST (50.8 ± 2.6 nM) was 10-fold lower than that of the commercially obtained AChE, indicating that AFEST had higher sensitivity. Its sensitivity for the other OPs was lower than AChE. To enhance the sensitivity of AFEST to OPs, site-directed mutations were introduced in the cap domain of AFEST. The sensitivity of mutant N44S/S48V was enhanced toward all seven OPs compared to the wild-type and was higher than AChE for four OPs, including paraoxon (3.3 ± 0.01 nM), dichlorvos (28.0 ± 0.6 nM), profenofos (43.0 ± 1.0 nM), and diazinon (3.0 ± 0.2 nM). The half-lives of AFEST and the mutant N44S/S48V at 37 °C were over 15 d. The interactions between the enzymes and select OPs were investigated by molecular docking. The results demonstrated that AFEST and the mutant N44S/S48V have the potential to be biosensor for OP detection.     

Carbohydrate carbamate coated onto microporous silica: Application to chiral analysis of commercial pharmaceutical drugs

The enantioselectivity of a series of chiral analytes was examined on tris-(3,5-dimethylphenyl carbamate) coated onto microporous APS-silica. The effects of the nature of the carbohydrate, the weight ratio of carbamate/support and the type of alcohol present in the mobile phase were investigated. Among the chiral analytes examined, propanolol showed an extremely good resolution. The resolution of tetramisole was optimized and the method developed was shown to be suitable for analysis of the drug in commercial tablets and in a suspension. © 1996 Wiley-Liss, Inc.     

Rat heart is a site of leptin production and action

Leptin, the 16-kDa peptide hormone product of the ob gene, is produced primarily by adipocytes and was initially thought to exert its effects exclusively through actions on the hypothalamus via distinct leptin receptors termed OB-R. However, recent data show that leptin is produced elsewhere and that receptors are present in many other tissues. Using real-time PCR, we determined whether leptin and its receptors are present in the rat heart and demonstrated regional distribution patterns and gender differences as well as the effect of ischemia and reperfusion. Gene expression of leptin and its receptors (OB-Ra, OB-Rb, and OB-Re) was identified in myocytes and whole heart homogenates from all regions of the heart of male and female rats, with the highest abundance in left and right atria of male and female rats, respectively. No differences in regional distribution of OB-R were evident in male rat hearts. In female rats, expression was highest in right atria for all three isoforms and was significantly greater than in male rats. Ischemia and reperfusion significantly downregulated leptin and OB-R expression, although this was more pronounced in male rat hearts. Leptin release in the coronary effluent was also detected using ELISA, although this was generally unaffected by global ischemia and reperfusion. Our results demonstrate for the first time the presence of the leptin system, including the peptide and its receptors, in all regions of the rat heart. In view of emerging evidence for cardiac effects of leptin, it is proposed that the heart is a target for leptin action and that the peptide modulates function through a paracrine- or autocrine-dependent manner.     

Fluorescence method for determining the mechanism and speed of action of surface-active drugs on yeast cells

New antifungal agents are needed to treat life-threatening fungal infections, particularly with the development of resistance. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a time-dependent drug exposure assay that allows us to rapidly investigate the mechanism of surface-active antifungal drug action. The assay uses a multidrug pump-deficient strain of Saccharomyces cerevisiae and the potentiometric dye 3,3'-dipropylthiacarbocyanine iodide [diS-C?(3)] and can assess whether cells are depolarized, hyperpolarized, or permeabilized by drug exposure. In this work, we investigated the mechanisms of action of five surface-active compounds: SDS, nystatin, amphotericin B, octenidine dihydrochloride, and benzalkonium chloride. The diS-C?(3) time-dependent drug exposure assay can be used to identify the mechanisms of action of a wide range of drugs. It is a fast and cost-effective method for screening drugs to determine their lowest effective concentrations.     

Strategies to improve plasma half life time of peptide and protein drugs

Summary. Due to the obvious advantages of long-acting peptide and protein drugs, strategies to prolong plasma half life time of such compounds are highly on demand. Short plasma half life times are commonly due to fast renal clearance as well as to enzymatic degradation occurring during systemic circulation. Modifications of the peptide/protein can lead to prolonged plasma half life times. By shortening the overall amino acid amount of somatostatin and replacing l-analogue amino acids with d-amino acids, plasma half life time of the derivate octreotide was 1.5 hours in comparison to only few minutes of somatostatin. A PEG_2,40 K conjugate of INF-α-2b exhibited a 330-fold prolonged plasma half life time compared to the native protein. It was the aim of this review to provide an overview of possible strategies to prolong plasma half life time such as modification of N- and C-terminus or PEGylation as well as methods to evaluate the effectiveness of drug modifications. Furthermore, fundamental data about most important proteolytic enzymes of human blood, liver and kidney as well as their cleavage specificity and inhibitors for them are provided in order to predict enzymatic cleavage of peptide and protein drugs during systemic circulation.     

Propulsive action of a snake pushing against a single site: Its combined analysis

The simultaneous use of electromyography (EMG), strain gauges, and cinematography show that the capacity of continuous displacement from a single peg is based on the following: sequential activity of the tested muscles from front to rear; activity restricted to the short portion of the body in contact with the peg; alternate action of the muscle longissimus dorsi on the two sides, the transition between one side to the other occurring at the site of contact with the peg; unilateral activity of the muscle supracostalis ventralis responsible for a bulging against the peg; a great stability in the direction of the resultant force, which makes only a small angle with the directio of the motion.     

Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase

The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The healing skin wound: a novel site of action of the chemokine C10

To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. For this purpose we generated a subtractive cDNA library from normal mouse back skin and 1-day full-thickness excisional wounds. One of the differentially expressed genes encodes the chemokine C10. Using Northern blotting, RNase protection assay and Western blotting, we confirmed the injury-induced expression of C10 at the mRNA and protein level. Maximal levels of C10 mRNA and protein were seen at day 1 after wounding, and expression levels subsequently declined. In situ hybridization and immunohistochemistry revealed expression of C10 in macrophages of the clot and the granulation tissue as well as in keratinocytes of the epidermis and the hair follicles at the wound edge. Since C10 is a potent chemoattractant for macrophages, our results suggest that this chemokine contributes to the strong macrophage influx observed in the healing skin wound.