Preparation and in vivo evaluation of sodium alginate - poly (vinyl alcohol) electrospun nanofibers of forskolin for glaucoma treatment.

The present investigation is aiming to prepare Sodium Alginate (SA) - Poly (vinyl alcohol) (PVA) nanofibrous mats of Forskolin (FSK) for ocular delivery to treat the glaucoma. Nanofibers of SA: PVA (1:0.25) load with ß- cyclodextrin- FSK solid dispersion were successfully prepared by an electrospinning technique. Eight formulations were Prepared and evaluated for drug content, scanning electron microscopy, degree of swelling, drug release and In Vivo Intra ocular pressure (IOP) reduction studies. The morphological studies revealed that average diameter of prepare nano fibers were decreased for formulations with low polymer concentration. Less diameter and uniform surface was observed for formulations F4 and F8 which are prepared under applied voltage 20kV, Capillary tip-to-Collector distance 15cm conditions. From the degree of swelling studies, it was observed that thinner the nanofiber mats, the greater the degree of swelling. The burst release within one hour was seen for F1 to F4 formulations whereas up to 90 min for F5 to F8 formulations. Release kinetic studies revealed that release of drug from the Nanofibrous mats have followed zero order kinetics. The results of in vivo IOP reduction studies suggested that FSK loaded Nanofibrous mats formulation (F4) produced a significant and controlled reduction in IOP throughout 45h.

Nano-emulsions as vehicles for topical delivery of forskolin.

Two O/W forskolin-loaded nano-emulsions (0.075% wt.) based on medium chain triglycerides (MCT) and stabilized by a nonionic surfactant (Polysorbate 80 or Polysorbate 40) were studied as forskolin delivery systems. The nano-emulsions were prepared by the PIC method. The mean droplet size of the nano-emulsions with Polysorbate 80 and Polysorbate 40 with oil/surfactant (O/S) ratios of 20/80 and 80% water concentration, measured by Dynamic Light Scattering (DLS), was of 118 nm and 111 nm, respectively. Stability of the formulations, as assessed by light backscattering for 24 h, showed that both nano-emulsions were stable at 25°C. Studies of forskolin in vitro skin permeation from the nano-emulsions and from a triglyceride solution were carried out at 32°C, using Franz-type diffusion cells. A mixture of PBS/ethanol (60/40 v/v) was used as a receptor solution. The highest flux and permeability coefficient was obtained for the system stabilized with Polysorbate 80 (6.91±0.75 µg · cm-2·h-1 and 9.21 · 10-3±1.00 · 10-3 cm · h-1, respectively) but no significant differences were observed with the flux and permeability coefficient value of forskolin dissolved in oil. The obtained results showed that the nano-emulsions developed in this study could be used as effective carriers for topical administration of forskolin.

Transdermal delivery of forskolin from emulsions differing in droplet size.

The skin permeation of forskolin, a diterpene isolated from Coleus forsholii, was studied using oil in water (O/W) emulsions as delivery formulations and also an oil solution for comparative purposes. Two forskolin-loaded emulsions of water/Brij 72:Symperonic A7/Miglyol 812:Isohexadecane, at 0.075 wt% forskolin concentration were prepared with the same composition and only differing in droplet size (0.38 µm and 10 µm). The emulsions showed high kinetic stability at 25 °C. In vitro study of forskolin penetration through human skin was carried out using the MicroettePlus(®) system. The concentration of the active in the receptor solution (i.e. ethanol/phosphate buffer 40/60, v/v) was analyzed by high performance liquid chromatography with UV detection. The obtained results showed that forskolin permeation from the emulsions and the oil solution, through human skin, was very high (up to 72.10%), and no effect of droplet size was observed.

Intestinal permeability of forskolin by in situ single pass perfusion in rats.

The intestinal permeability of forskolin was investigated using a single pass intestinal perfusion (SPIP) technique in rats. SPIP was performed in different intestinal segments (duodenum, jejunum, ileum, and colon) with three concentrations of forskolin (11.90, 29.75, and 59.90 µg/mL). The investigations of adsorption and stability were performed to ensure that the disappearance of forskolin from the perfusate was due to intestinal absorption. The results of the SPIP study indicated that forskolin could be absorbed in all segments of the intestine. The effective permeability (P (eff)) of forskolin was in the range of drugs with high intestinal permeability. The P (eff) was highest in the duodenum as compared to other intestinal segments. The decreases of P (eff) in the duodenum and ileum at the highest forskolin concentration suggested a saturable transport process. The addition of verapamil, a P-glycoprotein inhibitor, significantly enhanced the permeability of forskolin across the rat jejunum. The absorbed fraction of dissolved forskolin after oral administration in humans was estimated to be 100 % calculated from rat P (eff). In conclusion, dissolved forskolin can be absorbed readily in the intestine. The low aqueous solubility of forskolin might be a crucial factor for its poor oral bioavailability.

A sensitive and specific HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of isoforskolin in beagle dogs.

A sensitive and specific high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method has been developed and validated for the determination of isoforskolin in canine plasma. Liquid-liquid extraction was used to extract isoforskolin and the internal standard (I.S.) eplerenone from canine plasma. The chromatographic separation was performed on an Agela Venusil XBP Phenyl column with an isocratic mobile phase consisting of methanol-2mM ammonium acetate-formic acid (62:38:0.1, v/v/v), pumped at 0.35 mL/min. Isoforskolin and I.S. were detected at m/z 433.4→373.3 and m/z 415.3→163.5 in positive ion and multiple reaction monitoring (MRM) mode, respectively. The standard curves were linear over the concentration range of 0.1-200 ng/mL (r>0.99). The intra- and inter-batch accuracy values for isoforskolin at four concentrations were 90.2-108.3% and 97.8-106.6%, respectively. The RSDs were less than 6.0%. The mean extraction recoveries of isoforskolin and I.S. were 97.0 and 88.4%, respectively. The method was successfully applied to the pharmacokinetic study after an intravenous administration of isoforskolin in beagle dogs.

Design and evaluation of thermoreversible in situ gelling system of forskolin for the treatment of glaucoma.

The contact time of a vehicle on the cornea is of utmost importance for ophthalmic drug delivery. The poor bioavailability and therefore poor therapeutic response exhibited by the conventional ophthalmic solutions due to pre-corneal elimination of a drug may be overcome by the use of the in situ gel forming systems, which upon instillation as drops into the eye, undergo a sol-gel transition in the cul-de-sac. The purpose of this work was to develop an ophthalmic delivery system of the forskolin, based on the concept of temperature activated in situ gelation. Poloxamer 407 (Pluronic F-127) is a block copolymer made of poly (oxy ethylene) and poly (oxy propylene). The sol-gel transition is induced by an increase in temperature; however, it depends on the concentration of the polymer and presence of other additives. The developed formulations were therapeutically efficacious (on albino New Zealand rabbit model); reducing intra ocular pressure (IOP) for 12 hours and showed sol-gel phase transition (gelling) temperature of 22 degrees C and sustained drug release 72% +/- 0.056% in vitro behavior over a period of 4 h.

Segmental heterogeneity of electrogenic secretions in human ascending colon and rectum.

AIMS: We have attempted to ascertain putative segmental differences in the secretory responses of the human ascending colon and rectum. METHODS: From the mucosal biopsy samples of two segments, the short-circuit current (I(sc)) and tissue resistance (R(te)) were compared under control conditions, as well as after the induction of secretion, using a modified Ussing chamber. We also performed semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to detect and quantify transport proteins. RESULTS: The spontaneous I(sc) in the ascending colon was found to be greater than that in the rectum (P<0.01), whereas isobutylmethylxanthine/forskolin and carbachol (CCh) induced a greater rise in I(sc) in the rectum than in the ascending colon (P<0.05). When coupled with indomethacin pretreatment, the increase in Delta I(sc) after the addition of CCh and forskolin was significant as compared to that observed without pretreatment (P<0.05). However, in the rectum, the secretory response to CCh and forskolin was abolished to a significant degree by indomethacin (P<0.05). Moreover, these indomethacin-induced changes were reversed by the addition of PGE2. Upon semiquantitative RT-PCR analysis, the amounts of cystic fibrosis transmembrane regulator, KCNQ1, and CLCA1 mRNAs were not found to be different between the two segments. CONCLUSION: There was a clear segmental heterogeneity with regard to electrogenic secretion in the human colon, and this difference can be explained by differences in the ascending colon and rectum.

Cannabinoid CB1 receptor-mediated modulation of evoked dopamine release and of adenylyl cyclase activity in the human neocortex.

1. The present study investigated the binding characteristics of various ligands to cannabinoid CB(1) receptors in human neocortex and amygdala. In addition, the functionality of CB(1) receptors in the human neocortex was assessed by examining the effects of CB(1) receptor ligands on evoked [(3)H]-dopamine (DA) release in superfused brain slices and on synaptosomal cAMP accumulation. 2. Saturation-binding assays in human neocortical and amygdala synaptosomes using a radiolabelled cannabinoid receptor agonist ([(3)H]-CP55.940) revealed pK(d) values of 8.96 and 8.63, respectively. The numbers of binding sites (B(max)) were 3.99 and 2.67 pmol (mg protein)(-1), respectively. 3. Various cannabinoid receptor ligands inhibited [(3)H]-CP55.940 binding with rank order potencies corresponding to those of previous studies in animal tissues. 4. Electrically evoked [(3)H]-DA release from human neocortical slices was inhibited by CP55.940 (IC(50) 6.76 nm, I(max) 65%) and strongly enhanced by the cannabinoid receptor antagonist AM251. However, [(3)H]-DA release was not influenced in rat neocortex. In human tissue, the estimated endocannabinoid concentration in the biophase of the release-modulating CB(1) receptors was 1.07 nm, expressed in CP55.940 units. 5. K(+)-evoked [(3)H]-DA release in the presence of tetrodotoxin (TTX) was strongly inhibited by CP55.940 in humans, but not in rats. 6. In human tissue, CP55.940 inhibited forskolin-stimulated cAMP accumulation (IC(50) 20.89 nm, I(max) 35%). AM251 blocked this effect and per se increased forskolin-stimulated cAMP accumulation by approximately 20%. 7. In conclusion, cannabinoids modulate [(3)H]-DA release and adenylyl cyclase activity in the human neocortex. CB(1) receptors are located on dopaminergic nerve terminals and seem to be tonically activated by endocannabinoids.

Pharmacokinetics and a simulation model of colforsin daropate, new forskolin derivative inotropic vasodilator, in patients undergoing coronary artery bypass grafting.

Colforsin daropate, a water-soluble forskolin derivative, is an adenyl cyclase activator with positive inotropic and vasodilatory effects that are useful in the treatment of ventricular dysfunction. We investigated the pharmacokinetics of colforsin daropate in cardiac surgery patients and performed simulations to determine the dosage necessary to maintain an effective plasma concentration following cardiopulmonary bypass. In six patients undergoing coronary artery bypass graft, colforsin daropate (0.01mgkg(-1)) was administered immediately after separation from cardiopulmonary bypass. Arterial blood was sampled over the next 16h and plasma concentrations of colforsin daropate and its initial active metabolite were determined by gas-chromatography. Extended nonlinear least-squares regression was used to fit a three-compartment model to each patient's data. Distribution half-life (t(1/2alpha)) was 3.9+/-1.1min, metabolic half-life (t(1/2beta)) was 1.9+/-0.7h, and elimination half-life (t(1/2gamma)) was 95.3+/-15.2h. Central-compartment volume was 591.0+/-42.8mlkg(-1), volume distribution was 2689.2+/-450.6mlkg(-1), and elimination clearance was 27.7+/-14.7mlkg(-1)min(-1). In the pharmacokinetic simulation model, 0.5, 0.75, and 1.0microgkg(-1)min(-1) continuous infusion of colforsin daropate produce effective concentration (5-10ngml(-1)) within 30, 20, and 10min, respectively following administration. An initial active metabolite of decreased rapidly to less than 1.0ngml(-1) within the first 10min.A colforsin daropate infusion of 0.7-1.0microgkg(-1)min(-1) for 10-20min followed by 0.5microgkg(-1)min(-1) continuous infusion is recommended to produce an effective concentration (5-10ngml(-1)) within 10-20min and to maintain a therapeutic concentration throughout the administration period after cardiopulmonary bypass.

Endothelin-1 limits vascular smooth muscle beta-adrenergic receptor sensitivity by a PKC-dependent pathway.

Endothelin-1 reduces the chronotropic and inotropic effects of the beta-adrenoceptor agonist isoproterenol in rabbit isolated atria. Vascular interactions between endothelin-1 and isoproterenol have not been reported. Rings of the rabbit aorta without endothelium were mounted on myographs to measure isometric tension. Vessels were precontracted to similar levels with phenylephrine (30 micromol/L) or endothelin-1 (30 nmol/L). Relaxation to isoproterenol and forskolin were obtained. Vascular sensitivity (pD2) to isoproterenol was not different in the presence of endothelin-1 (7.6 +/- 0.3; n = 13) and phenylephrine (7.5 +/- 0.3; n = 11). The maximal relaxation (Emax) however, was doubled (P < 0.05) by endothelin-1 (42 +/- 5%), as compared with phenylephrine (23 +/- 4%). In the presence of endothelin-1, chelerythrine (protein kinase C inhibitor; 10 micromol/L) increased (P < 0.05) vascular sensitivity to isoproterenol (8.6 +/- 0.4, n = 7), but had no influence on the Emax. In contrast, in the presence of phenylephrine, pD2 was unaffected by chelerythrine, whereas the Emax to isoproterenol was increased (P < 0.05; 50 +/- 4%, n = 8). Vascular sensitivity and Emax to forskolin were similar in the presence of endothelin-1 and phenylephrine. In conclusion, endothelin-1 reduces vascular sensitivity to isoproterenol in a PKC-dependent pathway. The permissive effect of endothelin-1 appears to directly target the beta-adrenoceptor/G protein complex upstream of adenylate cyclase.

Positive regulation of GABA(B) receptors dually coupled to cyclic AMP by the allosteric agent CGP7930.

The ability of 2,6 Di-tert-butyl-4-(-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), a positive allosteric modulator of GABA(B) receptors, to regulate GABA(B) receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat brain was investigated. In olfactory bulb granule cell layer and in frontal cortex, CGP7930 potentiated the stimulatory effects of (-)-baclofen and gamma-aminobutyric acid (GABA) on basal and corticotropin-releasing hormone-stimulated adenylyl cyclase activities, respectively. In these stimulatory responses, CGP7930 enhanced both agonist potencies and maximal effects. When GABA(B) receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity of frontal cortex was examined, CGP7930 increased the agonist potencies but failed to affect the maximal effect of (-)-baclofen and modestly increased that of GABA. Similar results were obtained for the inhibition of Ca(2+)/calmodulin-stimulated adenylyl cyclase in striatum and cerebellum. Western blot analysis of each membrane preparation showed the presence of GABA(B2) receptor subunit, a putative site of action of CGP7930. These data indicate that CGP7930 positively modulates brain GABA(B) receptors coupled to either stimulation or inhibition of cyclic AMP signalling.

Two-photon excitation imaging of pancreatic islets with various fluorescent probes.

Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca(2+)-sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca(2+) measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membrane-impermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution.

[Cardiovascular effects of colforsin daropate hydrochloride, a novel drug for the treatment of acute heart failure].

Colforsin daropate hydrochloride (COL) is a water-soluble forskolin derivative for the treatment of acute heart failure. COL, like forskolin, stimulated adenylate cyclase (AC) directly and produced pharmacologic activities accompanied by the increase in cellular cAMP. COL was different from forskolin in water-solubility, duration of action, BBB permeability, oral activity and AC-subtype selectivity. COL was a inodilator with positive inotropic and vasodilator effects and was effective on a beta-receptor desensitized-heart model in which the effects of beta-agonists and PDE inhibitors were attenuated. COL improved cardiac function in some heart failure models. In the clinical studies, COL improved hemodynamics, subjective and objective symptoms of heart failure patients, and was also effective in the catecholamine-resistant heart failure patients. COL is a first clinically available adenylate cyclase activator. Further information from the post-marketing-surveillance will provide information that will enable more adequate usage of this drug.

Distribution of adenylate cyclase in the brain of Apteronotus leptorhynchus as revealed by forskolin binding.

An antibody directed against an isoform of the rat regulatory subunit of protein kinase A and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 53 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in fish brains. The 53-kDa band was enriched in fish forebrain. [3H]Forskolin binding was used as a marker for the distribution of adenylate cyclase. [3H]Forskolin binding was nearly completely displaced by excess cold forskolin; specific [3H]forskolin binding sites were heterogenously distributed with relatively high densities in some gray matter regions and low densities in fiber tracts. A high density of [3H]forskolin binding sites was found in the dorsal forebrain with lower densities in most ventral forebrain nuclei. Moderate binding densities were observed in the preoptic and hypothalamic areas with the exception of the nucleus tuberis anterior, which had high levels. The thalamus and midbrain had low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was low. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal and ventral molecular layers, which contain feedback inputs; the cellular layers of the ELL had low binding densities.

Changes in protein kinase C and adenylate cyclase in the temporal lobe from subjects with schizophrenia.

Changes in G-protein linked neurotransmitter receptors have been reported in a number of regions of the brain of schizophrenic subjects. These changes, if functional, could cause a change in proteins such as protein kinase C (PKC) and adenylate cyclase (AC) which are important components of the G-protein linked second messenger cascades. We therefore used autoradiography to measure the distribution and density of [3H]phorbol ester binding to PKC and [3H]forskolin binding to AC in tissue obtained at autopsy from schizophrenic and non-schizophrenic subjects (Controls). There were significant decreases in the density of PKC in the parahippocampal gyrus (687 +/- 60 vs. 885 +/- 51 fmol/mg TE; mean +/- SEM; p < 0.01) and in AC in the dentate gyrus (75 +/- 4.9 vs. 92 +/- 6.5, p < 0.05) from the schizophrenic subjects. These data could indicate that changes in neurotransmitter receptors in the hippocampus from subjects with schizophrenia could have resulted in a change in their associated second messenger systems.

Potential antidepressant properties of forskolin and a novel water-soluble forskolin (NKH477) in the forced swimming test.

The mechanisms of the antidepressant activity of forskolin and a novel water soluble forskolin analog (NKH477) were studied using the forced swimming method in rats. Forskolin (0.01-0.1 mg/kg) and NKH477 (0.01-0.1 mg/kg) dose-dependently decreased ratings of immobility, with effects similar to those of amitriptyline treatment. The maximum effects of forskolin and NKH477 were observed at 0.01 mg/kg dose which is 150 more times potent than that (15 mg/kg) of amitriptyline. At a high dose (1.0 mg/kg) of forskolin and NKH477, the duration of immobility was returned to control levels. Forskolin and NKH477 did not influence the spontaneous locomotor activity at intraperitoneal injection doses from 0.01 to 1 mg/kg. Furthermore chronic administration with NKH477 at oral dose from 0.5 to 1.5 mg/kg significantly decreases the duration of immobility. These data indicate that both forskolin and NKH477 have strong antidepressive potency, consistent with the hypothesis that elevation of the cAMP cascade system may have an important role in antidepressive effects.

Neurally mediated cardiac effects of forskolin in conscious dogs.

Because major cardiovascular disease states are characterized by defects in adenylyl cyclase regulation, it becomes important to understand the mechanisms by which adenylyl cyclase activators affect inotropy and chronotropy in intact conscious animals. Accordingly, we examined the inotropic and chronotropic responses to forskolin in 11 normal conscious, chronically instrumented dogs and 3 dogs with ventricular denervation (VD). Left ventricular first derivative of pressure (LV dP/dt) increased by 96 +/- 7%, P < 0.05, in response to forskolin (50 nmol.kg-1.min-1) in normal dogs and by significantly less, 52 +/- 14%, in VD dogs. Circulating norepinephrine (NE) levels increased similarly in both groups (from 226 +/- 18 to 389 +/- 33 pg/ml in normal dogs, from 177 +/- 23 to 329 +/- 71 pg/ml in VD dogs). In the presence of ganglionic blockade, the increase in LV dP/dt in response to forskolin was reduced (+62 +/- 4%) in normal dogs but was unchanged in VD dogs (+52 +/- 12%). Ganglionic blockade abolished the increase in circulating NE levels in both groups. Increases in heart rate in the presence of ganglionic blockade (+54 +/- 6 beats/min) were less than in the presence of atropine alone (+92 +/- 10 beats/min). Notably, the LV dP/dt and heart rate responses to forskolin were further attenuated by beta-adrenergic receptor blockade in the presence and absence of ganglionic blockade. Morphine also attenuated the increases in both LV dP/dt and plasma NE in response to forskolin. Increases in LV dP/dt in response to NKH-477 (30 micrograms/kg), a water-soluble forskolin derivative, were similar before and after ganglionic blockade (+63 +/- 8 and +51 +/- 10%, respectively). However, in vitro experiments in LV sarcolemmal membrane preparations demonstrated that stimulation of adenylyl cyclase by forskolin and NKH-477 was not affected by beta-adrenergic receptor blockade. These results indicate that in conscious dogs, inotropic and chronotropic effects of forskolin are not only due to direct activation of adenylyl cyclase, but the effects also are mediated by neural mechanisms and potentiated by the prevailing level of sympathetic tone.

The pharmacodynamics of 6-(3-dimethylaminopropionyl)forskolin and a possible metabolite in beagles.

A specific gas chromatography/mass spectroscopy method with a detection limit of 0.1 ng/mL was developed for the measurement of 6-(3-dimethylaminopropionyl)forskolin (1) in beagle plasma. Using this method, plasma concentrations of 1 in beagles given pharmacologically effective intravenous doses of 1.HCl were determined. The observed maximal plasma concentrations rapidly decreased with time, and half-lives of the alpha-phases were < 9 min. Pharmacological effects of 1 on the cardiovascular parameters were simultaneously evaluated in one of the studies. Decreases of the pharmacological effects were slower than decreases in plasma concentration of 1. In addition, 6-(3-methylaminopropionyl)forskolin (N-monodemethyl 1), an expected initial metabolite of 1, was prepared and found to be as pharmacologically active as 1 in beagles. These results and others strongly suggest that a metabolite(s) of 1 contributes to the pharmacological effects of 1 in beagles.

Immunomodulation by an adenylate cyclase activator, NKH477, in vivo and vitro.

Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger which modulates T cell function. NKH477 is a direct adenylate cyclase activator derived from forskolin and now under clinical investigation as a positive inotropic agent. While the immunosuppressive effects of forskolin on lymphocytes have been reported, little is known about its effects in vivo. In this study, we investigated whether NKH477 has immunosuppressive effects in mice, namely on cardiac allograft survival, and on the generation of cytotoxic T lymphocytes (CTL), T cell proliferation in mixed lymphocyte reaction (MLR), and production of interleukin-2 (IL-2) in MLR and in mitogen response. We assessed the effects of standard immunosuppressant cyclosporin A (CsA) on IL-2 production and on allograft survival to estimate the intensity of rejection in this acute rejection model. Saline-treated C57BL/6 (H-2b) mice rejected DBA/2 (H-2d) cardiac allografts with a median graft survival time of 10 days. In contrast, median graft survival was prolonged to 12 and 15 days in mice treated with NKH477 at 1 and 3 mg/kg/day, respectively (P < 0.01 vs control). The equivalent dose of CsA (40 mg/kg/day) to the maintenance dose after clinical cardiac transplantation prolonged median graft survival time to 15.5 days, indicating that high dose of NKH477 was as efficacious as lower dose of CsA. Addition of NKH477 to the culture medium suppressed the generation of CTL, T cell proliferation in MLR, and production of IL-2 in MLR and in mitogen response. These results suggest that NKH477 exerts a beneficial effect on murine cardiac allograft survival by modulating T cell function.

Kinetics of drug delivery to the arterial wall via polyurethane-coated removable nitinol stent: comparative study of two drugs.

Polymer-coated removable stents were used to deliver 14C-labeled etretinate and 3H-labeled forskolin to the vessel wall in 31 New Zealand White rabbits to study their kinetics. Stents loaded with etretinate (n = 8) and forskolin (n = 14) were implanted in the rabbit carotid arteries, and the animals were euthanized at different time intervals. Drug levels were measured in the media and adventitia of the stented segment, in distant tissues, and in blood. In four rabbits, forskolin-loaded stents were percutaneously retrieved 2 hr after implantation in the carotid artery, and the tissue and blood levels were determined 2 and 24 hr after retrieval. In seven rabbits etretinate-loaded stents were retrieved 72 hr after implantation in abdominal aorta, and drug levels were measured in the tissues and blood immediately after and at 1 and 4 days after retrieval. Levels of etretinate in the vessel wall peaked at 24 hr (250 ng/mg) and remained high up to 72 hr (185 ng/mg) after stent placement. Levels of forskolin peaked within 2 hr of stent placement (135 ng/mg) and rapidly declined to 4.9 ng/mg at 24 hr with the stent in situ. About 50% (1.4 mg) of the original etretinate remained in the stent at 72 hr compared to about 5% (0.08 mg) of forskolin at 24 hr. Ratio of peak drug levels in the vessel wall to those in the blood was 6,000 for etretinate and 780 for forskolin. (ABSTRACT TRUNCATED AT 250 WORDS)