Intratracheally Administered Peptide-Modified Lipid Admixture Containing Fasudil and/or DETA NONOate Ameliorates Various Pathologies of Pulmonary Arterial Hypertension.

This study examined the therapeutic potential of a combination therapy using fasudil, a Rho-kinase inhibitor, and DETA NONOate (DN), a nitric oxide donor, delivered as a lipid admixture modified with a cyclic homing peptide known as CAR (CAR-lipid mixture) for the treatment of pulmonary arterial hypertension (PAH). CAR-lipid mixtures were initially prepared via a thin-film hydration method and then combined with fasudil, DN, or a mixture of both. The therapeutic efficacy of this drug-laden lipid mixture was evaluated in a Sugen/Hypoxia (Su/Hx) rat model of PAH by measuring RV systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), Fulton indices, and assessing right ventricular (RV) functions, as well as evaluating pulmonary vascular morphology. Rats that received no treatment exhibited increases in RVSP, mPAP, Fulton indices, and changes in RV functional parameters. However, the treatment with the CAR-lipid mixture containing either fasudil or DN or a combination of both led to a decline in mPAP, RVSP, and Fulton indices compared to saline-treated rats. Similarly, rats that received these treatments showed concurrent improvement in various echocardiographic parameters such as pulmonary acceleration time (PAT), tricuspid annular plane systolic excursion (TAPSE), and ventricular free wall thickness (RVFWT). A significant decrease in the wall thickness of pulmonary arteries larger than 100 µm was observed with the combination therapy. The findings reveal that fasudil, DN, and their combination in a CAR-modified lipid mixture improved pulmonary hemodynamics, RV functions, and pathological alterations in the pulmonary vasculature. This study underscores the potential of combination therapy and targeted drug delivery in PAH treatment, laying the groundwork for future investigations into the optimization of these treatments, their long-term safety and efficacy, and the underlying mechanism of action of the proposed therapy.

Catheterization of Pulmonary and Carotid Arteries for Concurrent Measurement of Mean Pulmonary and Systemic Arterial Pressure in Rat Models of Pulmonary Arterial Hypertension.

Pulmonary hypertension (PH) is a group of pulmonary vascular disorders in which mean pulmonary arterial pressure (mPAP) becomes abnormally high because of various pathological conditions, including remodeling of the pulmonary arteries, lung and heart disorders, or congenital conditions. Various animal models, including mouse and rat models, have been used to recapitulate elevated mPAP observed in PH patients. However, the measurement and recording of mPAP and mean systemic arterial pressure (mSAP) in small animals require microsurgical procedures and a sophisticated data acquisition system. In this paper, we describe the surgical procedures for right heart catheterizations (RHC) to measure mPAP in rats. We also explain the catheterization of the carotid artery for simultaneous measurement of mPAP and mSAP using the PowerLab Data Acquisition system. We enumerate the surgical steps involved in exposing the jugular vein and the carotid artery for catheterizing these two blood vessels. We list the tools used for microsurgery in rats, describe the methods for preparing catheters, and illustrate the process for inserting the catheters in the pulmonary and carotid arteries. Finally, we delineate the steps involved in the calibration and setup of the PowerLab system for recording both mPAP and mSAP. This is the first protocol wherein we meticulously explain the surgical procedures for RHC in rats and the recording of mPAP and mSAP. We believe this protocol will be essential for PH research. Investigators with little training in animal handling can reproduce this microsurgical procedure for RHC in rats and measure mPAP and mSAP in rat models of PH. Further, this protocol is likely to help master RHC in rats that are performed for other conditions, such as heart failure, congenital heart disease, heart valve disorders, and heart transplantation.

An Overview of Organ-on-a-Chip Models for Recapitulating Human Pulmonary Vascular Diseases.

Traditionally, animal models have been used for recapitulating human physiology and for studying the pathological basis of many diseases affecting humankind. Indeed, over the centuries, animal models helped advance our understanding of the biology and pathology of drug therapy for humans. However, with the advent of genomics and pharmacogenomics, we now know that conventional models cannot accurately capture the pathological conditions and biological processes in humans, although humans share many physiological and anatomical features with many animals [1-3]. Species to species variation have raised concerns about the validity and suitability of animal models for studying human conditions. Over the past decade, the development and advances in microfabrication and biomaterials have spurred the growth in micro-engineered tissue and organ models (organs-on-a-chip, OoC) as alternatives to animal and cellular models [4]. This state-of-the-art technology has been used to emulate human physiology for investigating multitudes of cellular and biomolecular processes implicated in the pathological basis of disease (Fig. 13.1) [4]. Because of their tremendous potential, OoC-based models have been listed as one of the top 10 emerging technologies in the 2016 World Economic Forum [2].

Emerging biologics for the treatment of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare pulmonary vascular disorder, wherein mean systemic arterial pressure (mPAP) becomes abnormally high because of aberrant changes in various proliferative and inflammatory signalling pathways of pulmonary arterial cells. Currently used anti-PAH drugs chiefly target the vasodilatory and vasoconstrictive pathways. However, an imbalance between bone morphogenetic protein receptor type II (BMPRII) and transforming growth factor beta (TGF-ß) pathways is also implicated in PAH predisposition and pathogenesis. Compared to currently used PAH drugs, various biologics have shown promise as PAH therapeutics that elicit their therapeutic actions akin to endogenous proteins. Biologics that have thus far been explored as PAH therapeutics include monoclonal antibodies, recombinant proteins, engineered cells, and nucleic acids. Because of their similarity with naturally occurring proteins and high binding affinity, biologics are more potent and effective and produce fewer side effects when compared with small molecule drugs. However, biologics also suffer from the limitations of producing immunogenic adverse effects. This review describes various emerging and promising biologics targeting the proliferative/apoptotic and vasodilatory pathways involved in PAH pathogenesis. Here, we have discussed sotatercept, a TGF-ß ligand trap, which is reported to reverse vascular remodelling and reduce PVR with an improved 6-minute walk distance (6-MWDT). We also elaborated on other biologics including BMP9 ligand and anti-gremlin1 antibody, anti-OPG antibody, and getagozumab monoclonal antibody and cell-based therapies. Overall, recent literature suggests that biologics hold excellent promise as a safe and effective alternative to currently used PAH therapeutics.

An evolving perspective on novel modified release drug delivery systems for inhalational therapy.

INTRODUCTION: Drugs delivered via the lungs are predominantly used to treat various respiratory disorders, including asthma, chronic obstructive pulmonary diseases, respiratory tract infections and lung cancers, and pulmonary vascular diseases such as pulmonary hypertension. To treat respiratory diseases, targeted, modified or controlled release inhalation formulations are desirable for improved patient compliance and superior therapeutic outcome. AREAS COVERED: This review summarizes the important factors that have an impact on the inhalable modified release formulation approaches with a focus toward various formulation strategies, including dissolution rate-controlled systems, drug complexes, site-specific delivery, drug-polymer conjugates, and drug-polymer matrix systems, lipid matrix particles, nanosystems, and formulations that can bypass clearance via mucociliary system and alveolar macrophages. EXPERT OPINION: Inhaled modified release formulations can potentially reduce dosing frequency by extending drug's residence time in the lungs. However, inhalable modified or controlled release drug delivery systems remain unexplored and underdeveloped from the commercialization perspective. This review paper addresses the current state-of-the-art of inhaled controlled release formulations, elaborates on the avenues for developing newer technologies for formulating various drugs with tailored release profiles after inhalational delivery and explains the challenges associated with translational feasibility of modified release inhalable formulations.

A Protocol for Fabrication and on-Chip Cell Culture to Recreate PAH-Afflicted Pulmonary Artery on a Microfluidic Device.

Pulmonary arterial hypertension (PAH) is a rare pulmonary vascular disease that affects people of all ethnic origins and age groups including newborns. In PAH, pulmonary arteries and arterioles undergo a series of pathological changes including remodeling of the entire pulmonary vasculatures and extracellular matrices, mis-localized growth of pulmonary arterial cells, and development of glomeruloid-like lesions called plexiform lesions. Traditionally, various animal and cellular models have been used to understand PAH pathophysiology, investigate sex-disparity in PAH and monitor therapeutic efficacy of PAH medications. However, traditional models can only partially capture various pathological features of PAH, and they are not adaptable to combinatorial study design for deciphering intricately intertwined complex cellular processes implicated in PAH pathogenesis. While many microfluidic chip-based models are currently available for major diseases, no such disease-on-a-device model is available for PAH, an under investigated disease. In the absence of any chip-based models of PAH, we recently proposed a five-channel polydimethylsiloxane (PDMS)-based microfluidic device that can emulate major pathological features of PAH. However, our proposed model can make a bigger impact on the PAH field only when the larger scientific community engaged in PAH research can fabricate the device and develop the model in their laboratory settings. With this goal in mind, in this study, we have described the detailed methodologies for fabrication and development of the PAH chip model including a thorough explanation of scientific principles for various steps for chip fabrication, a detailed list of reagents, tools and equipment along with their source and catalogue numbers, description of laboratory setup, and cautionary notes. Finally, we explained the methodologies for on-chip cell seeding and application of this model for studying PAH pathophysiology. We believe investigators with little or no training in microfluidic chip fabrication can fabricate this eminently novel PAH-on-a-chip model. As such, this study will have a far-reaching impact on understanding PAH pathophysiology, unravelling the biological mystery associated with sexual dimorphism in PAH, and developing PAH therapy based on patient sex and age.

Co-amorphous Drug Delivery Systems: a Review of Physical Stability, In Vitro and In Vivo Performance.

Over the past few decades, co-amorphous solids have been used as a promising approach for delivering poorly water-soluble drugs. Co-amorphous solids, comprising pharmacologically relevant drug substances or excipients, improve physical stability, solubility, dissolution, and bioavailability compared with single amorphous ingredients. In this review, we have summarized recent advances in physical stability and in vitro and in vivo performances of co-amorphous solids. We have highlighted the role of molar ratio, molecular interaction, and mobility that affects the physical stability of co-amorphous solids. This review delves deep as to how co-amorphous solids affect the physicochemical properties in vitro and in vivo. We also described the challenges to the formulation of co-amorphous solids. A better understanding of the mechanisms of the physical stability, in vitro and in vivo performance of co-amorphous solids, and proper selection of the co-former is likely to expedite the development of robust co-amorphous-based pharmaceutical formulations and can address the challenges associated with the delivery of poorly soluble drugs.

Physical stability and dissolution behaviors of amorphous pharmaceutical solids: Role of surface and interface effects.

Amorphous pharmaceutical solids (APS) are single- or multi-component systems in which drugs exist in high-energy states with long-range disordered molecular packing. APSs have become one of the most effective and widely used pharmaceutical delivery approaches for poorly water-soluble drugs in the last several decades. Considerable efforts have been made to investigate the physical stability and dissolution behaviors of APSs, however, the underlying mechanisms remain imperfectly understood. Recent studies reveal that surface and interface properties of APSs could strongly affect the physical stability and dissolution behaviors. This paper provides a comprehensive overview of recent studies focusing on the physical stability and dissolution behaviors of APSs from both surface and interface perspectives. We highlight the role of surface or interface properties in nucleation, crystal growth, phase separation, dissolution, and supersaturation. Meanwhile, the challenges and scope of research on surface and interface properties in the future are also briefly discussed. This review contributes to a better understanding of the surface- and interface-facilitated processes, which will provide more efficient and rational guidance for the design of APSs.

Multilayer Soft Photolithography Fabrication of Microfluidic Devices Using a Custom-Built Wafer-Scale PDMS Slab Aligner and Cost-Efficient Equipment.

We present a robust, low-cost fabrication method for implementation in multilayer soft photolithography to create a PDMS microfluidic chip with features possessing multiple height levels. This fabrication method requires neither a cleanroom facility nor an expensive UV exposure machine. The central part of the method stays on the alignment of numerous PDMS slabs on a wafer-scale instead of applying an alignment for a photomask positioned right above a prior exposure layer using a sophisticated mask aligner. We used a manual XYZR stage attached to a vacuum tweezer to manipulate the top PDMS slab. The bottom PDMS slab sat on a rotational stage to conveniently align with the top part. The movement of the two slabs was observed by a monocular scope with a coaxial light source. As an illustration of the potential of this system for fast and low-cost multilayer microfluidic device production, we demonstrate the microfabrication of a 3D microfluidic chaotic mixer. A discussion on another alternative method for the fabrication of multiple height levels is also presented, namely the micromilling approach.

Multicellular Cell Seeding on a Chip: New Design and Optimization towards Commercialization.

This paper shows both experimental and in-depth theoretical studies (including simulations and analytical solutions) on a microfluidic platform to optimize its design and use for 3D multicellular co-culture applications, e.g., creating a tissue-on-chip model for investigating diseases such as pulmonary arterial hypertension (PAH). A tissue microfluidic chip usually has more than two channels to seed cells and supply media. These channels are often separated by barriers made of micro-posts. The optimization for the structures of these micro-posts and their spacing distances is not considered previously, especially for the aspects of rapid and cost-efficient fabrication toward scaling up and commercialization. Our experimental and theoretical (COMSOL simulations and analytical solutions) results showed the followings: (i) The cell seeding was performed successfully for this platform when the pressure drops across the two posts were significantly larger than those across the channel width. The circular posts can be used in the position of hexagonal or other shapes. (ii) In this work, circular posts are fabricated and used for the first time. They offer an excellent barrier effect, i.e., prevent the liquid and gel from migrating from one channel to another. (iii) As for rapid and cost-efficient production, our computer-aided manufacturing (CAM) simulation confirms that circular-post fabrication is much easier and more rapid than hexagonal posts when utilizing micro-machining techniques, e.g., micro-milling for creating the master mold, i.e., the shim for polymer injection molding. The findings open up a possibility for rapid, cost-efficient, large-scale fabrication of the tissue chips using micro-milling instead of expensive clean-room (soft) lithography techniques, hence enhancing the production of biochips via thermoplastic polymer injection molding and realizing commercialization.

3D Printing in Solid Dosage Forms and Organ-on-Chip Applications.

3D printing (3DP) can serve not only as an excellent platform for producing solid dosage forms tailored to individualized dosing regimens but can also be used as a tool for creating a suitable 3D model for drug screening, sensing, testing and organ-on-chip applications. Several new technologies have been developed to convert the conventional dosing regimen into personalized medicine for the past decade. With the approval of Spritam, the first pharmaceutical formulation produced by 3DP technology, this technology has caught the attention of pharmaceutical researchers worldwide. Consistent efforts are being made to improvise the process and mitigate other shortcomings such as restricted excipient choice, time constraints, industrial production constraints, and overall cost. The objective of this review is to provide an overview of the 3DP process, its types, types of material used, and the pros and cons of each technique in the application of not only creating solid dosage forms but also producing a 3D model for sensing, testing, and screening of the substances. The application of producing a model for the biosensing and screening of drugs besides the creation of the drug itself, offers a complete loop of application for 3DP in pharmaceutics.

Epigenetic Mechanisms as Emerging Therapeutic Targets and Microfluidic Chips Application in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a disease that progress over time and is defined as an increase in pulmonary arterial pressure and pulmonary vascular resistance that frequently leads to right-ventricular (RV) failure and death. Epigenetic modifications comprising DNA methylation, histone remodeling, and noncoding RNAs (ncRNAs) have been established to govern chromatin structure and transcriptional responses in various cell types during disease development. However, dysregulation of these epigenetic mechanisms has not yet been explored in detail in the pathology of pulmonary arterial hypertension and its progression with vascular remodeling and right-heart failure (RHF). Targeting epigenetic regulators including histone methylation, acetylation, or miRNAs offers many possible candidates for drug discovery and will no doubt be a tempting area to explore for PAH therapies. This review focuses on studies in epigenetic mechanisms including the writers, the readers, and the erasers of epigenetic marks and targeting epigenetic regulators or modifiers for treatment of PAH and its complications described as RHF. Data analyses from experimental cell models and animal induced PAH models have demonstrated that significant changes in the expression levels of multiple epigenetics modifiers such as HDMs, HDACs, sirtuins (Sirt1 and Sirt3), and BRD4 correlate strongly with proliferation, apoptosis, inflammation, and fibrosis linked to the pathological vascular remodeling during PAH development. The reversible characteristics of protein methylation and acetylation can be applied for exploring small-molecule modulators such as valproic acid (HDAC inhibitor) or resveratrol (Sirt1 activator) in different preclinical models for treatment of diseases including PAH and RHF. This review also presents to the readers the application of microfluidic devices to study sex differences in PAH pathophysiology, as well as for epigenetic analysis.

Design, synthesis and biological evaluations of a long-acting, hypoxia-activated prodrug of fasudil, a ROCK inhibitor, to reduce its systemic side-effects.

ROCK, one of the downstream regulators of Rho, controls actomyosin cytoskeleton organization, stress fiber formation, smooth muscle contraction, and cell migration. ROCK plays an important role in the pathologies of cerebral and coronary vasospasm, hypertension, cancer, and arteriosclerosis. Pharmacological-induced systemic inhibition of ROCK affects both the pathological and physiological functions of Rho-kinase, resulting in hypotension, increased heart rate, decreased lymphocyte count, and eventually cardiovascular collapse. To overcome the adverse effects of systemic ROCK inhibition, we developed a bioreductive prodrug of a ROCK inhibitor, fasudil, that functions selectively under hypoxic conditions. By masking fasudil's active site with a bioreductive 4-nitrobenzyl group, we synthesized a prodrug of fasudil that is inactive in normoxia. Reduction of the protecting group initiated by hypoxia reveals an electron-donating substituent that leads to fragmentation of the parent molecule. Under normoxia the fasudil prodrug displayed significantly reduced activity against ROCK compared to its parent compound, but under severe hypoxia the prodrug was highly effective in suppressing ROCK activity. Under hypoxia the prodrug elicited an antiproliferative effect on disease-afflicted pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. The prodrug displayed a long plasma half-life, remained inactive in the blood, and produced no drop in systemic blood pressure when compared with fasudil-treated controls. Due to its selective nature, our hypoxia-activated fasudil prodrug could be used to treat diseases where tissue-hypoxia or hypoxic cells are the pathological basis of the disease.

Delayed Exercise-induced Upregulation of Angiogenic Proteins and Recovery of Motor Function after Photothrombotic Stroke in Mice.

Treatments promoting post-stroke functional recovery continue to be an unmet therapeutic problem with physical rehabilitation being the most reproduced intervention in preclinical and clinical studies. Unfortunately, physiotherapy is typically effective at high intensity and early after stroke - requirements that are hardly attainable by stroke survivors. The aim of this study was to directly evaluate and compare the dose-dependent effect of delayed physical rehabilitation (daily 5 h or overnight voluntary wheel running; initiated on post-stroke day 7 and continuing through day 21) on recovery of motor function in the mouse photothrombotic model of ischemic stroke and correlate it with angiogenic potential of the brain. Our observations indicate that overnight but not 5 h access to running wheels facilitates recovery of motor function in mice in grid-walking test. Western blotting and immunofluorescence microscopy experiments evaluating the expression of angiogenesis-associated proteins VEGFR2, doppel and PDGFRß in the peri-infarct and corresponding contralateral motor cortices indicate substantial upregulation of these proteins (≥2-fold) in the infarct core and surrounding cerebral cortex in the overnight running mice on post-stroke day 21. These findings indicate that there is a dose-dependent relationship between the extent of voluntary exercise, motor recovery and expression of angiogenesis-associated proteins in this expert-recommended mouse ischemic stroke model. Notably, our observations also point out to enhanced angiogenesis and presence of pericytes within the infarct core region during the chronic phase of stroke, suggesting a potential contribution of this tissue area in the mechanisms governing post-stroke functional recovery.

Newer insights into the pathobiological and pharmacological basis of the sex disparity in patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) affects more women than men, although affected females tend to survive longer than affected males. This sex disparity in PAH is postulated to stem from the diverse roles of sex hormones in disease etiology. In animal models, estrogens appear to be implicated not only in pathologic remodeling of pulmonary arteries, but also in protection against right ventricular (RV) hypertrophy. In contrast, the male sex hormone testosterone is associated with reduced survival in male animals, where it is associated with increased RV mass, volume, and fibrosis. However, it also has a vasodilatory effect on pulmonary arteries. Furthermore, patients of both sexes show varying degrees of response to current therapies for PAH. As such, there are many gaps and contradictions regarding PAH development, progression, and therapeutic interventions in male versus female patients. Many of these questions remain unanswered, which may be due in part to lack of effective experimental models that can consistently reproduce PAH pulmonary microenvironments in their sex-specific forms. This review article summarizes the roles of estrogens and related sex hormones, immunological and genetical differences, and the benefits and limitations of existing experimental tools to fill in gaps in our understanding of the sex-based variation in PAH development and progression. Finally, we highlight the potential of a new tissue chip-based model mimicking PAH-afflicted male and female pulmonary arteries to study the sex-based differences in PAH and to develop personalized therapies based on patient sex and responsiveness to existing and new drugs.

Pulmonary-arterial-hypertension (PAH)-on-a-chip: fabrication, validation and application.

Currently used animal and cellular models for pulmonary arterial hypertension (PAH) only partially recapitulate its pathophysiology in humans and are thus inadequate in reproducing the hallmarks of the disease, inconsistent in portraying the sex-disparity, and unyielding to combinatorial study designs. Here we sought to deploy the ingenuity of microengineering in developing and validating a tissue chip model for human PAH. We designed and fabricated a microfluidic device to emulate the luminal, intimal, medial, adventitial, and perivascular layers of a pulmonary artery. By growing three types of pulmonary arterial cells (PACs)-endothelial, smooth muscle, and adventitial cells, we recreated the PAH pathophysiology on the device. Diseased (PAH) PACs, when grown on the chips, moved of out their designated layers and created phenomena similar to the major pathologies of human PAH: intimal thickening, muscularization, and arterial remodeling and show an endothelial to mesenchymal transition. Flow-induced stress caused control cells, grown on the chips, to undergo morphological changes and elicit arterial remodeling. Our data also suggest that the newly developed chips can be used to elucidate the sex disparity in PAH and to study the therapeutic efficacy of existing and investigational anti-PAH drugs. We believe this miniaturized device can be deployed for testing various prevailing and new hypotheses regarding the pathobiology and drug therapy in human PAH.

Critical physicochemical attributes of chitosan nanoparticles admixed lactose-PEG 3000 microparticles in pulmonary inhalation.

Chitosan nanoparticles are exhalation prone and agglomerative to pulmonary inhalation. Blending nanoparticles with lactose microparticles (∼5 µm) could mutually reduce their agglomeration through surface adsorption phenomenon. The chitosan nanoparticles of varying size, size distribution, zeta potential, crystallinity, shape and surface roughness were prepared by spray drying technique as a function of chitosan, surfactant and processing conditions. Lactose-polyethylene glycol 3000 (PEG3000) microparticles were similarly prepared. The chitosan nanoparticles, physically blended with fine lactose-PEG3000 microparticles, exhibited a comparable inhalation performance with the commercial dry powder inhaler products (fine particle fraction between 20% and 30%). Cascade impactor analysis indicated that the aerosolization and inhalation performance of chitosan nanoparticles was promoted by their higher zeta potential and circularity, and larger size attributes of which led to reduced inter-nanoparticulate aggregation and favored nanoparticles interacting with lactose-PEG3000 micropaticles that aided their delivery into deep and peripheral lungs.

Newer approaches and novel drugs for inhalational therapy for pulmonary arterial hypertension.

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of small pulmonary arteries leading to increased pulmonary arterial pressure. Existing treatments acts to normalize vascular tone via three signaling pathways: the prostacyclin, the endothelin-1, and the nitric oxide. Although over the past 20 years, there has been considerable progress in terms of treatments for PAH, the disease still remains incurable with a disappointing prognosis.Areas covered: This review summarizes the pathophysiology of PAH, the advantages and disadvantages of the inhalation route, and assess the relative advantages various inhaled therapies for PAH. The recent studies concerning the development of controlled-release drug delivery systems loaded with available anti-PAH drugs have also been summarized.Expert opinion: The main obstacles of current pharmacotherapies of PAH are their short half-life, stability, and formulations, resulting in reducing the efficacy and increasing systemic side effects and unknown pathogenesis of PAH. The pulmonary route has been proposed for delivering anti-PAH drugs to overcome the shortcomings. However, the application of approved inhaled anti-PAH drugs is limited. Inhalational delivery of controlled-release nanoformulations can overcome these restrictions. Extensive studies are required to develop safe and effective drug delivery systems for PAH patients.

Statistical optimization and validation of a novel ultra-performance liquid chromatography method for estimation of nintedanib in rat and human plasma.

Aim: A high throughput ultra-performance liquid chromatography (UPLC)-ultraviolet method for quantification of nintedanib in rat and human plasma was developed and optimized using chemometrical approach. Method: Design of experiment and multivariate statistical approach was used for definition of optimized method. Final separation was performed using protein precipitation method on ACQUITY HSS T3 C18 column in isocratic mode using potassium phosphate buffer (pH 7.5): acetonitrile. Results: Method was validated as per US-FDA guidelines linearly from 15-750 ng/ml. All quality control samples showed <15% relative standard deviation for precision and 85-115% accuracy along with >98% extraction recovery. Conclusion: The developed method is easily applicable in determining pharmacokinetic parameters in preclinical subjects along with successful implementation for quantification in human plasma samples.

CAR, a Homing Peptide, Prolongs Pulmonary Preferential Vasodilation by Increasing Pulmonary Retention and Reducing Systemic Absorption of Liposomal Fasudil.

Here, we sought to elucidate the role of CAR (a cyclic peptide) in the accumulation and distribution of fasudil, a drug for pulmonary arterial hypertension (PAH), in rat lungs and in producing pulmonary specific vasodilation in PAH rats. As such, we prepared liposomes of fasudil and CAR-conjugated liposomal fasudil and assessed the liposomes for CAR conjugation, physical properties, entrapment efficiencies, in vitro release profiles, and stabilities upon incubation in cell culture media, storage, and aerosolization. We also studied the cellular uptake of fasudil in different formulations, quantified heparan sulfate (HS) in pulmonary arterial smooth muscle cells (PASMCs), and investigated the distribution of the liposomes in the lungs of PAH rats. We assessed the drug accumulation in a close and recirculating isolated perfused rat lung model and studied the pharmacokinetics and pharmacological efficacy of the drug and formulations in Sugen/hypoxia-induced PAH rats. The entrapment efficiency of the liposomal fasudil was 95.5 ± 4.5%, and the cumulative release was 93.95 ± 6.22%. The uptake of CAR liposomes by pulmonary arterial cells and their distribution and accumulation in the lungs were much greater than those of no-CAR-liposomes. CAR-induced increase in the cellular uptake was associated with an increase in HS expression by rat PAH-PASMCs. CAR, when conjugated with liposomal fasudil and given via an intratracheal instillation, extended the elimination half-life of the drug by four-fold compared with fasudil-in-no-CAR-liposomes given via the same route. CAR-conjugated liposomal fasudil, as opposed to fasudil-in-no-CAR-liposomes and CAR pretreatment followed by fasudil-in-no-CAR-liposomes, reduced the mean pulmonary arterial pressure by 40-50% for 6 h, without affecting the mean systemic arterial pressure. On the whole, this study suggests that CAR aids in concentrating the drug in the lungs, increasing the cellular uptake, extending the half-life of fasudil, and eliciting a pulmonary-specific vasodilation when the peptide remains conjugated on the liposomal surface, but not when CAR is given as a pretreatment or alone as an admixture with the drug.