Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Nitric oxide inhibits the release of norepinephrine and dopamine from the medial basal hypothalamus of the rat.

Previous research indicates that norepinephrine and dopamine stimulate release of luteinizing hormone (LH)-releasing hormone (LHRH), which then reaches the adenohypophysis via the hypophyseal portal vessels to release LH. Norepinephrine exerts its effect via alpha 1-adrenergic receptors, which stimulate the release of nitric oxide (NO) from nitricoxidergic (NOergic) neurons in the medial basal hypothalamus (MBH). The NO activates guanylate cyclase and cyclooxygenase, thereby inducing release of LHRH into the hypophyseal portal vessels. We tested the hypothesis that these two catecholamines modulate NO release by local feedback. MBH explants were incubated in the presence of sodium nitroprusside (NP), a releaser of NO, and the effect on release of catecholamines was determined. NP inhibited release of norepinephrine. Basal release was increased by incubation of the tissue with the NO scavenger hemoglobin (20 micrograms/ml). Hemoglobin also blocked the inhibitory effect of NP. In the presence of high-potassium (40 mM) medium to depolarize cell membranes, norepinephrine release was increased by a factor of 3, and this was significantly inhibited by NP. Hemoglobin again produced a further increase in norepinephrine release and also blocked the action of NP. When constitutive NO synthase was inhibited by the competitive inhibitor NG-monomethyl-L-arginine (NMMA) at 300 microM, basal release of norepinephrine was increased, as was potassium-evoked release, and this was associated in the latter instance with a decrease in tissue concentration, presumably because synthesis did not keep up with the increased release in the presence of NMMA. The results were very similar with dopamine, except that reduction of potassium-evoked dopamine release by NP was not significant. However, the increase following incubation with hemoglobin was significant, and hemoglobin, when incubated with NP, caused a significant elevation in dopamine release above that with NP alone. In this case, NP increased tissue concentration of dopamine along with inhibiting release, suggesting that synthesis continued, thereby raising the tissue concentration in the face of diminished release. When the tissue was incubated with NP plus hemoglobin, which caused an increase in release above that obtained with NP alone, the tissue concentration decreased significantly compared with that in the absence of hemoglobin, indicating that, with increased release, release exceeded synthesis, causing a fall in tissue concentration. When NO synthase was blocked by NMMA, the release of dopamine, under either basal or potassium-evoked conditions, was increased. Again, in the latter instance the tissue concentration declined significantly, presumably because synthesis did not match release. Therefore, the results were very similar with both catecholamines and indicate that NO acts to suppress release of both amines. Since both catecholamines activate the release of LHRH, the inhibition of their release by NO serves as an ultra-short-loop negative feedback by which NO inhibits the release of the catecholamines, thereby reducing the activation of the NOergic neurons and decreasing the release of LHRH. This may be an important means for terminating the pulses of release of LHRH, which generate the pulsatile release of LH that stimulates gonadal function in both male and female mammals.

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.

The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide.

In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L-arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity.

Nitric oxide mediates the increase in local cerebral blood flow during focal seizures.

The role of nitric oxide (NO) in the increase in local cerebral blood flow (LCBF) elicited by focal cortical epileptic seizures was investigated in anesthetized adult rats. Seizures were induced by topical bicuculline methiodide applied through two cranial windows drilled over homotopic sites of the frontal cortex, and LCBF was measured by quantitative autoradiography by using 4-iodo[N-methyl-14C]antipyrine. Superfusion of an inhibitor of NO synthase, N omega-nitro-L-arginine (NA; 1 mM), for 45 min abolished the increase of LCBF induced by topical bicuculline methiodide (10 mM) [164 +/- 18 ml/100 g per min in the artificial cerebrospinal fluid (aCSF)-superfused side and 104 +/- 12 ml/100 g per ml in the NA-superfused side; P < 0.005]. This effect was reversed by coapplication of an excess of L-arginine substrate (10 mM) (218 +/- 22 ml/100 g per min in the aCSF-superfused side and 183 +/- 31 ml/100 g per min in the NA + L-Arg-superfused side) but not by 10 mM D-arginine, a stereoisomer with poor affinity for NO synthase (193 +/- 17 ml/100 g per min in the aCSF-superfused side and 139 +/- 21 ml/100 g per min in the NA + D-Arg-superfused side; P < 0.005). Superfusion of the guanylyl cyclase inhibitor methylene blue attenuated the LCBF increase elicited by topical bicuculline methiodide by 25% +/- 16% (P < 0.05). The present findings suggest that NO is the mediator of the vasodilation in response to focal epileptic seizures.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

Nitric oxide synthase inhibition in sepsis? Lessons learned from large-animal studies

Nitric Oxide (NO) plays a controversial role in the pathophysiology of sepsis and septic shock. Its vasodilatory effects are well known, but it also has pro- and antiinflammatory properties, assumes crucial importance in antimicrobial host defense, may act as an oxidant as well as an antioxidant, and is said to be a vital poison for the immune and inflammatory network. Large amounts of NO and peroxynitrite are responsible for hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis, and ultimately multiorgan failure. Therefore, NO synthase (NOS) inhibitors were developed to reverse the deleterious effects of NO. Studies using these compounds have not met with uniform success however, and a trial using the nonselective NOS inhibitor N-G-methyl-L-arginine hydrochloride was terminated prematurely because of increased mortality in the treatment arm despite improved shock resolution. Thus, the issue of NOS inhibition in sepsis remains a matter of debate. Several publications have emphasized the differences concerning clinical applicability of data obtained from unresuscitated, hypodynamic rodent models using a pretreatment approach versus resuscitated, hyperdynamic models in high-order species using posttreatment approaches. Therefore, the present review focuses on clinically relevant large-animal studies of endotoxin or living bacteria-induced, hyperdynamic models of sepsis that integrate standard day-today care resuscitative measures.

Renal endothelial dysfunction and impaired autoregulation after ischemia-reperfusion injury result from excess nitric oxide

Endothelial dysfunction in ischemic acute renal failure (IARF) has been attributed to both direct endothelial injury and to altered endothelial nitric oxide synthase ( eNOS) activity, with either maximal upregulation of eNOS or inhibition of eNOS by excess nitric oxide ( NO) derived from iNOS. We investigated renal endothelial dysfunction in kidneys from Sprague-Dawley rats by assessing autoregulation and endothelium-dependent vasorelaxation 24 h after unilateral ( U) or bilateral ( B) renal artery occlusion for 30 (U30, B30) or 60 min (U60, B60) and in sham-operated controls. Although renal failure was induced in all degrees of ischemia, neither endothelial dysfunction nor altered facilitation of autoregulation by 75 pM angiotensin II was detected in U30, U60, or B30 kidneys. Baseline and angiotensin II-facilitated autoregulation were impaired, methacholine EC50 was increased, and endothelium-derived hyperpolarizing factor ( EDHF) activity was preserved in B60 kidneys. Increasing angiotensin II concentration restored autoregulation and increased renal vascular resistance ( RVR) in B60 kidneys; this facilitated autoregulation, and the increase in RVR was abolished by 100 mu M furosemide. Autoregulation was enhanced by N-omega-nitro-L-arginine methyl ester. Peri-ischemic inhibition of inducible NOS ameliorated renal failure but did not prevent endothelial dysfunction or impaired autoregulation. There was no significant structural injury to the afferent arterioles with ischemia. These results suggest that tubuloglomerular feedback is preserved in IARF but that excess NO and probably EDHF produce endothelial dysfunction and antagonize autoregulation. The threshold for injury-producing, detectable endothelial dysfunction was higher than for the loss of glomerular filtration rate. Arteriolar endothelial dysfunction after prolonged IARF is predominantly functional rather than structural.

Nitric oxide in the rat gastrointestinal tract

This study concerns the nature of nitric oxide synthase (NOS) and the role of nitric oxide (NO) in the rat gastrointestinal tract. The major objectives were (i) to characterise NOS isoforms in the gastric glandular mucosa, (ii) to localise NOS isoforms in the rat gastric glandular mucosa, (iii) to investigate the role of NO in carbachol-stimulated gastric mucus secretion, (iv) to investigate the nature of NOS and small intestine. Immunoblotting was performed using polyclonal antisera raised against two peptides found in the rat brain NOS sequence and commercial monoclonal antibodies directed against neuronal and endothelial isoforms of NOS. A160kDa band was detected in brain and gastric mucosal samples with antibodies and antisera directed against neuronal NOS sequences, and a 140kDa band was detected in gastric mucosal samples using an anti-endothelial NOS antibody. An intense 160kDa neuronal NOS band was detected in a high-density fraction of gastric mucosal cells separated on a Percoll gradient. Detection of neuronal NOS by a carboxyl-terminal antiserum in samples of brain, but not of gastric mucosa, could be blocked by the peptide (20g/ml) against which the antibody was raised. After affinity purification, recognition of gastric mucosal NOS was blocked by peptide. Particulate neuronal NOS was found in the brain by immunoblotting while 94% of gastric mucosal enzyme was soluble. Gastric mucosal endothelial NOS was 95% particulate. 95% of NOS activity in the gastric mucosa was due to neuronal NOS. Paraformaldehyde- and acetone-fixed gastric mucosal sections were subject to immunocytochemistry using the above antibodies. Neuronal NOS was localised to the surface mucosal epithelial cells while endothelial NOS was associated with microvessels at the base of the mucosa and to larger vessels in the submucosa. Intragastric administration of carbachol or 16, 16-dimethyl prostaglandin E2 increased the thickness of the rat gastric mucus layer. The NOS inhibitor NG-nitro-L-arginine methyl ester dose-dependently, and selectively, prevented the stimulatory effect of carbachol. Ca2+-independent NOS activity in rat ileal, jejunal and colonic muscle was increased after LPS induction. Ca2+-dependent activity was not affected. Distribution of inducible NOS protein paralleled Ca2+ -independent activity. LPS treatment did not affect the content of neuronal NOS in colonic muscle.

Aspects of signal transduction in the gastrointestinal tract

This study was undertaken to increase knowledge of the mechanisms of inter- and intracellular signalling in the gastrointestinal tract. Specific aims were: to use cell lines to elucidate factors affecting growth of gastric cells, to investigate the distribution and aspects of function of isoforms of protein kinase C in a gastric cell line and in the rat gastrointestinal tract and to determine the presence and regulation of nitric oxide synthase in gastrointestinal tissues from the rat and in cell lines. The gastric cancer cell line HGT-1 was used to investigate control of growth. Increases in cell number were found to be dependent on the seeding density of the cells. In cells plated at low density insulin, epidermal growth factor and gastrin all increased cell number. Gastrin produced a bell-shaped dose response curve with a maximum activity at 5nM. No effect of gastrin was apparent in cells plated at high density. α and β isoforms of protein kinase C were found, by immunoblotting procedures, to be widespread in the gastrointestinal tract of the rat, but protein kinase Cε was confined to the gastric mucosa and gastrointestinal smooth muscle. HGT-1 cells contained protein kinase C α and ε but β or γ were not detected. Preincubation of HGT-1 cells for 24h with 1μM phorbol-12,13-dibutyrate down-regulated protein kinase C α but not ε. The inhibition by the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) of the histamine-stimulated increase in cAMP in HGT-1 cells was down regulated by phorbol-12,13-dibutyrate. Inhibition of histamine-stimulation of adenylate cyclase by TPA was Ca2+-dependent and inhibited by the addition of an antibody to protein kinase C α. A role for protein kinase C α in modulating the effect of histamine on adenylate cyclase in HGT-1 cells is suggested. No nitric oxide synthase activity was detected in the gastrointestinal cell lines HGT-l, MKN-45 or CaCo-2. Ca2+-dependent nitric oxide synthase activity was observed in the gastric mucosa and the gastrointestinal smooth muscle from stomach to colon. The gastric: mucosal enzyme was soluble and showed half-maximal activity at 400nM Ca2+. Pretreatment of rats with endotoxin (3mg/kg body weight) induced nitric oxide synthase activity in both jejunal, ileal and colonic mucosa and muscle. A major portion of the induced activity in ileal and colonic mucosa was Ca2+-independent. Nitric oxide synthase activity in a high-density fraction of gastric mucosal cells was inhibited in a dose-dependent fashion by L-nitroarginine, NG-monomethyl-L-arginine, trifluoperazine and L-canavanine (in descending order of potency). Preincubation with okadaic acid and addition of ATPlMg2+ to the homogenisation buffer inhibited enzyme activity, which implies that phosphorylation inhibits gastric mucosal nitric oxide synthase.

Microencapsulation strategies for islet transplantation

A variety of islet microencapsulation techniques have been investigated to establish which method provides the least occlusive barrier to net insulin release in vitro, and optimum biocompatibility for islet implantation in vivo. NMRI mouse islets have been microencapsulated with Na+ -alginate-poly-L-lysine (PLL)/poly-L-ornithine (PLO)-alginate, Ba2+ -alginate and agarose gels. Both free and microencapsulated islets responded to glucose challenge in static incubation and perifusion by significantly increasing their rate of insulin release and theophylline significantly potentiated the insulin response to glucose. While little insulin was released from microencapsulated islets after short term (2 hours) static incubation, significantly greater amounts were released in response to glucose challenge after extended (8-24 hours) incubation. However, insulin release from all types of microencapsulated islets was significantly reduced compared with free islets. Na+ -alginate-PLO-alginate microencapsulated islets were significantly more responsive to elevated glucose than Na+ -alginate-PLL-alginate microencapsulated islets, due to the enhanced porosity of PLO membranes. The outer alginate layer created a significant barrier to glucose/insulin exchange and reduced the insulin responsiveness of microencapsulated islets to glucose. Ba2+ -alginate membrane coated islets, generated by the density gradient method, were the most responsive to glucose challenge. Low concentrations of NG-monomethyl L-arginine (L-NMMA) had no significant effect on glucose stimulated insulin release from either free or microencapsulated islets. However, 1.0 mmol/1 L-NMMA significantly inhibited the insulin response of both free and microencapsulated islets to glucose challenge. In vivo work designed to evaluate the extent of pericapsular fibrosis after 28 days ip. and sc. implantation of microencapsulated islets into STZ-diabetic recipients, revealed that the inclusion of islets within microcapsules increased their immunogenicity and markedly increased the extent of pericapsular fibrosis. When the outer alginate layer was omitted from microcapsules, little or no pericapsular mononuclear cell deposition was observed. The subcutaneous site was not suitable for microencapsulated islet transplantation in NMRI recipient mice. Systemic immunosuppression using cyclosporin A was effective in preventing pericapsular mononuclear cell deposition, while L-NMMA loading into microcapsules had no significant effect on pericapsular fibrosis, although it did maintain the integrity of microencapsulated islets.

Use of amino acids as counterions improves the solubility of the BCS II model drug, indomethacin

The number of new chemical entities (NCE) is increasing every day after the introduction of combinatorial chemistry and high throughput screening to the drug discovery cycle. One third of these new compounds have aqueous solubility less than 20µg/mL [1]. Therefore, a great deal of interest has been forwarded to the salt formation technique to overcome solubility limitations. This study aims to improve the drug solubility of a Biopharmaceutical Classification System class II (BCS II) model drug (Indomethacin; IND) using basic amino acids (L-arginine, L-lysine and L-histidine) as counterions. Three new salts were prepared using freeze drying method and characterised by FT-IR spectroscopy, proton nuclear magnetic resonance ((1)HNMR), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). The effect of pH on IND solubility was also investigated using pH-solubility profile. Both arginine and lysine formed novel salts with IND, while histidine failed to dissociate the free acid and in turn no salt was formed. Arginine and lysine increased IND solubility by 10,000 and 2296 fold, respectively. An increase in dissolution rate was also observed for the novel salts. Since these new salts have improved IND solubility to that similar to BCS class I drugs, IND salts could be considered for possible waivers of bioequivalence.

Poly(glycerol adipate-co-ω-pentadecalactone) spray-dried microparticles as sustained release carriers for pulmonary delivery

Purpose: The aim of this work was to optimize biodegradable polyester poly(glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, microparticles as sustained release (SR) carriers for pulmonary drug delivery. Methods: Microparticles were produced by spray drying directly from double emulsion with and without dispersibility enhancers (L-arginine and L-leucine) (0.5-1.5%w/w) using sodium fluorescein (SF) as a model hydrophilic drug. Results: Spray-dried microparticles without dispersibility enhancers exhibited aggregated powders leading to low fine particle fraction (%FPF) (28.79±3.24), fine particle dose (FPD) (14.42±1.57 μg), with a mass median aerodynamic diameter (MMAD) 2.86±0.24 μm. However, L-leucine was significantly superior in enhancing the aerosolization performance ( L-arginine:%FPF 27.61±4.49-26.57±1.85; FPD 12.40±0.99-19.54±0.16 μg and MMAD 2.18±0.35-2. 98±0.25 μm, L-leucine:%FPF 36.90±3.6-43.38±5. 6; FPD 18.66±2.90-21.58±2.46 μg and MMAD 2.55±0.03-3. 68±0.12 μm). Incorporating L-leucine (1.5%w/w) reduced the burst release (24.04±3.87%) of SF compared to unmodified formulations (41.87±2.46%), with both undergoing a square root of time (Higuchi's pattern) dependent release. Comparing the toxicity profiles of PGA-co-PDL with L-leucine (1.5%w/w) (5 mg/ml) and poly(lactide-co-glycolide), (5 mg/ml) spray-dried microparticles in human bronchial epithelial 16HBE14o-cell lines, resulted in cell viability of 85.57±5.44 and 60.66±6.75%, respectively, after 72 h treatment. Conclusion:The above data suggest that PGA-co-PDL may be a useful polymer for preparing SR microparticle carriers, together with dispersibility enhancers, for pulmonary delivery. © Springer Science+Business Media, LLC 2011.