In vitro, ex vivo and in vivo methods of lung absorption for inhaled drugs.

The assessment and prediction of lung absorption and disposition are an increasingly essential preclinical task for successful discovery and product development of inhaled drugs for both local and systemic delivery. Hence, in vitro, ex vivo and in vivo preclinical methods of lung absorption continue to evolve with several technical, methodological and analytical refinements. As in vitro lung epithelial cell monolayer models, the air-liquid interface (ALI)-cultured Calu-3 cells have most frequently been used, but the NCI-H441 and hAELVi cells have now been proposed as the first immortalized human alveolar epithelial cells capable of forming highly-restricted monolayers.

Pharmacokinetic profile analyses for inhaled drugs in humans using the lung delivery and disposition model.

The kinetic clarification of lung disposition for inhaled drugs in humans via pharmacokinetic (PK) modeling aids in their development and regulation for systemic and local delivery, but remains challenging due to its multiplex nature. This study exercised our lung delivery and disposition kinetic model to derive the kinetic descriptors for the lung disposition of four drugs [calcitonin, tobramycin, ciprofloxacin and fluticasone propionate (FP)] inhaled via different inhalers from the published PK profile data. With the drug dose delivered to the lung (DTL) estimated from the corresponding γ-scintigraphy or in vivo predictive cascade impactor data, the model-based curve-fitting and statistical moment analyses derived the rate constants of lung absorption (k ) and non-absorptive disposition (k ).

A synthetic heparin mimetic that allosterically inhibits factor XIa and reduces thrombosis in vivo without enhanced risk of bleeding.

Background: Human factor XIa (FXIa) is an actively pursued target for development of safer anticoagulants. Our long-standing hypothesis has been that allosterism originating from heparin-binding site(s) on coagulation enzymes is a promising approach to yield safer agents.

Objectives: To develop a synthetic heparin mimetic as an inhibitor of FXIa so as to reduce clot formation in vivo but not carry high bleeding risk.

In Vivo-Relevant Transwell Dish-Based Dissolution Testing for Orally Inhaled Corticosteroid Products.

Purpose: To establish an in vivo-relevant Transwell dish-based dissolution test system for the "respirable" aerosols of inhaled corticosteroids (ICSs) using marketed inhaler products.

Methods: "Respirable" ≤ 5.8 or 6.

Salvianolic acid B as an anti-emphysema agent I: In vitro stimulation of lung cell proliferation and migration, and protection against lung cell death, and in vivo lung STAT3 activation and VEGF elevation.

Emphysema causes progressive and life-threatening alveolar structural destruction/loss, yet remains irreversible and incurable to date. Impaired vascular endothelial growth factor (VEGF) signaling has been proposed as a new pathogenic mechanism, and if so, VEGF recovery may enable reversal of emphysema. Thus, we hypothesized that salvianolic acid B (Sal-B), a polyphenol in traditional Chinese herbal danshen, is an alveolar structural recovery agent for emphysema by virtue of VEGF stimulation/elevation via activation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), as stimulating lung cell proliferation and migration, and protecting against lung cell death.

Salvianolic acid B as an anti-emphysema agent II: In vivo reversal activities in two rat models of emphysema.

Emphysema progressively destroys alveolar structures, leading to disability and death, yet remains irreversible and incurable to date. Impaired vascular endothelial growth factor (VEGF) signaling is an emerging pathogenic mechanism, thereby proposing a hypothesis that VEGF stimulation/elevation enables recovery from alveolar structural destruction and loss of emphysema. Our previous in vitro study identified that salvianolic acid B (Sal-B), a polyphenol of traditional Chinese herbal danshen, stimulated lung cell proliferation and migration, and protected against induced lung cell death, by virtue of signal transducer and activator of transcription 3 (STAT3) activation and VEGF stimulation/elevation.

In vitro lung epithelial cell transport and anti-interleukin-8 releasing activity of liposomal ciprofloxacin.

As a promising long-acting inhaled formulation, liposomal ciprofloxacin (Lipo-CPFX) was characterized in the in vitro human lung epithelial Calu-3 cell monolayer system, compared to ciprofloxacin in solution (CPFX). Its modulated absorptive transport and uptake, and sustained inhibitory activity against induced pro-inflammatory interleukin-8 (IL-8) release were examined. The absorptive transport and uptake kinetics for Lipo-CPFX and CPFX were determined at 0.

Sulfated dehydropolymer of caffeic acid: In vitro anti-lung cell death activity and in vivo intervention in emphysema induced by VEGF receptor blockade.

Induced lung cell death and impaired hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) signaling are proposed as a pathobiologic mechanism for alveolar structural destruction and loss in emphysema. We hypothesized that our sulfated dehydropolymer of caffeic acid, CDSO3, exerts anti-cell death activities and therapeutic interventions in emphysema by virtue of Fe chelation-based HIF-1α/VEGF stabilization and elevation. The Fe chelating activity was determined in the chromogenic ferrozine-Fe chelation inhibitory assay.

Development and characterization of a naturally derived lung extracellular matrix hydrogel.

The complexity and rapid clearance mechanisms of lung tissue make it difficult to develop effective treatments for many chronic pathologies. We are investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. The main objectives of this study include effective decellularization of porcine lung tissue, development of a hydrogel from the porcine ECM, and characterization of the material's composition, mechanical properties, and ability to support cellular growth.

Pulmonary delivery of anorectic oxyntomodulin in rats: food intake suppression, reduced body weight gain and pharmacokinetics.

Background: Oxyntomodulin (OXM1-37) is an anorectic gut-secreting peptide with a promise to treat obesity, but its needle-free delivery has yet to be successful.

Results: Pulmonary delivery of OXM1-37, but not its C-terminal octapeptides, caused dose-related, transient 4-6 h food intake suppression in rats. At 0.

Iloprost reverses established fibrosis in experimental right ventricular failure.

Prostacyclin and its analogues improve cardiac output and functional capacity in patients with pulmonary arterial hypertension (PAH); however, the underlying mechanism is not fully understood. We hypothesised that prostanoids have load-independent beneficial effects on the right ventricle (RV). Angio-obliterative PAH and RV failure were induced in rats with a single injection of SU5416 followed by 4 weeks of exposure to hypoxia.

Expansion of Antibacterial Spectrum of Muraymycins toward Pseudomonas aeruginosa.

It is urgent to develop novel anti-Pseudomonas agents that should also be active against multidrug resistant P. aeruginosa. Expanding the antibacterial spectrum of muraymycins toward P.

Sulfated caffeic acid dehydropolymer attenuates elastase and cigarette smoke extract-induced emphysema in rats: sustained activity and a need of pulmonary delivery.

Background: Although emphysema destroys alveolar structures progressively and causes death eventually, no drug has been discovered to prevent, intervene, and/or resolve this life-threatening disease. We recently reported that sulfated caffeic acid dehydropolymer CDSO3 is a novel potent triple-action inhibitor of elastolysis, oxidation, and inflammation in vitro, and therefore, a potential anti-emphysema agent. However, the in vivo therapeutic potency, duration and mode of actions, and effective route remain to be demonstrated.

Systemic delivery of biotherapeutics through the lung: opportunities and challenges for improved lung absorption.

The development of Exubera(®) (inhaled insulin) has paved the way for consideration of future inhaled biotherapeutic products for systemic delivery. This route of drug delivery favors highly potent small peptides without self-association and large proteins resistant to enzymatic degradation for high bioavailability, while likely resulting in transient therapeutic effects. Improved therapeutic benefits with a needle-free delivery, such as inhaled insulin, are also rational pursuits.

Selective CB2 agonists with anti-pruritic activity: discovery of potent and orally available bicyclic 2-pyridones.

The CB2 receptor has emerged as a potential target for the treatment of pruritus as well as pain without CB1-mediated side effects. We previously identified 2-pyridone derivatives 1 and 2 as potent CB2 agonists; however, this series of compounds was found to have unacceptable pharmacokinetic profiles with no significant effect in vivo. To improve these profiles, we performed further structural optimization of 1 and 2, which led to the discovery of bicyclic 2-pyridone 18e with improved CB2 affinity and selectivity over CB1.

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring.

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.

Design, synthesis, and binding mode prediction of 2-pyridone-based selective CB2 receptor agonists.

Selective CB2 agonists have the potential for treating pain without central CB1-mediated adverse effects. Screening efforts identified 1,2-dihydro-3-isoquinolone 1; however, this compound has the drawbacks of being difficult to synthesize with two asymmetric carbons on an isoquinolone scaffold and of having a highly lipophilic physicochemical property. To address these two major problems, we designed the 2-pyridone-based lead 15a, which showed moderate affinity for CB2.

Novel low molecular weight lignins as potential anti-emphysema agents: In vitro triple inhibitory activity against elastase, oxidation and inflammation.

No molecule has been found to be effective against emphysema to date primarily because of its complex pathogenesis that involves elastolysis, oxidation and inflammation. We here describe novel unsulfated or sulfated low molecular weight lignins (LMWLs) chemo-enzymatically prepared from 4-hydroxycinnamic acids monomers, as the first potent triple-action inhibitors of neutrophil elastase, oxidation and inflammation. The inhibitory potencies of three different cinnamic acid-based LMWLs were determined in vitro using chromogenic substrate hydrolysis assays, radical scavenging and lung cellular oxidative biomarker reduced glutathione (rGSH) assays, and lung cellular inflammatory biomarker NFκB and IL-8 assays, respectively.

Conformational restriction approach to β-secretase (BACE1) inhibitors: effect of a cyclopropane ring to induce an alternative binding mode.

Improvement of a drug's binding activity using the conformational restriction approach with sp³ hybridized carbon is becoming a key strategy in drug discovery. We applied this approach to BACE1 inhibitors and designed four stereoisomeric cyclopropane compounds in which the ethylene linker of a known amidine-type inhibitor 2 was replaced with chiral cyclopropane rings. The synthesis and biologic evaluation of these compounds revealed that the cis-(1S,2R) isomer 6 exhibited the most potent BACE1 inhibitory activity among them.

Determination of oxyntomodulin, an anorectic polypeptide, in rat plasma using 2D-LC-MS/MS coupled with ion pair chromatography.

Polypeptide therapeutics present a challenge for quantitative analysis when using immunoassays or recently, liquid chromatography-tandem mass spectrometry because of their structural similarities to endogenous proteins and peptides in plasma. In this assay, a Waters Oasis® mixed-mode anion exchange (MAX) microelution modified solid phase extraction (SPE) method coupled with two-dimensional reversed phase ion pair chromatography-tandem mass spectrometry was used for the validation and analysis of oxyntomodulin in rat plasma. Oxyntomodulin (OXM) and its isotope labeled internal standard were extracted from rat plasma and analyzed with a chromatographic run time of 8 min.

Synthesis of pacidamycin analogues via an Ugi-multicomponent reaction.

The second-generation synthesis of 3'-hydroxypacidamycin D (2) has been accomplished via an Ugi-four component reaction at a late stage of the synthesis. This approach provided ready access to a range of analogues including diastereomers of the diaminobutylic acid residue and hybrid-type analogues of mureidomycins. Biological evaluations of these analogues indicated that the stereochemistry at the diaminobutylic acid residue has a crucial impact on both the MraY biochemical inhibition and whole-cell antibacterial activity.

Total synthesis and biological evaluation of pacidamycin D and its 3'-hydroxy analogue.

Full details of the total synthesis of pacidamycin D (4) and its 3'-hydroxy analogue 32 are described. The chemically labile Z-oxyacyl enamide moiety is the most challenging chemical structure found in uridylpeptide natural products. Key elements of our approach to the synthesis of 4 include the efficient and stereocontrolled construction of the Z-oxyvinyl halides 6 and 7 and their copper-catalyzed cross-coupling with the tetrapeptide carboxamide 5, a thermally unstable compound containing a number of potentially reactive functional groups.

Total synthesis of pacidamycin D by Cu(I)-catalyzed oxy enamide formation.

The first total synthesis of pacidamycin D, which is expected to be a good candidate as an antibacterial agent against P. aeruginosa, is described. The key elements of our approach feature an efficient and stereocontrolled construction of the Z-oxyvinyl iodide and copper-catalyzed cross-coupling with the tetrapeptide carboxamide.

The particle has landed--characterizing the fate of inhaled pharmaceuticals.

Although there is a modest body of literature on the absorption of inhaled pharmaceuticals by normal lungs and some limited information from diseased lungs, there is still a surprising lack of mechanistic knowledge about the details of the processes involved. Where are molecules absorbed, what mechanisms are involved, how well are different lung regions penetrated, what are the determinants of metabolism and dissolution, and how best can one retard the clearance of molecules deposited in the lung or induce intracellular uptake by lung cells? Some general principles are evident: (1) small hydrophobic molecules are absorbed very fast (within tens of seconds) usually with little metabolism; (2) small hydrophilic molecules are absorbed fast (within tens of minutes), again with minimal metabolism; (3) very low water solubility of the drug can retard absorption; (4) peptides are rapidly absorbed but are significantly metabolized unless chemically protected against peptidases; (5) larger proteins are more slowly absorbed with variable bioavailabilities; and 6) insulin seems to be best absorbed distally in the lungs while certain antibodies appear to be preferentially absorbed in the upper airways. For local lung disease applications, and some systemic applications as well, many small molecules are absorbed much too fast for convenient and effective therapies.

In vitro aqueous fluid-capacity-limited dissolution testing of respirable aerosol drug particles generated from inhaler products.

Purpose: To develop a unique in vitro aqueous fluid-capacity-limited dissolution system for the kinetic assessment of respirable aerosol drug particles from inhaler products.

Methods: Aerosol particles of 5 inhaled corticosteroids (ICSs) from 7 inhaler products were collected in the aerodynamic 2.1-3.