Remigius U. Agu

In vitro and in vivo testing methods for respiratory drug delivery

Importance of the field: Successful respiratory drug delivery for local and systemic purposes is predicated on the availability of in vitro and in vivo methods for determining drug delivery and disposition following respiratory administration. Areas covered in this review: In this review, the relevance of new in vitro and in vivo methods for screening respiratory drug delivery is discussed. Specific topics covered include in vitro particle size characterization, in vitro dissolution test methods for respiratory formulations and in vitro respiratory absorption and disposition screening methods. Furthermore, in vivo respiratory dosing methods, in vivo respiratory aerosol deposition and drug absorption screening methods, and correlation between in vitro and in vivo methods are reviewed. What the reader will gain: After reading this article, the reader will have an enriched knowledge regarding the various in vitro and in vivo testing methods for respiratory drug delivery. Most importantly, this paper will make it possible for readers to appreciate the strengths and weaknesses of each test method, which in turn will assist them in selecting specific methods that suit their scientific needs. Take home message: New in vitro and in vivo methods for screening respiratory drug delivery are indispensible, especially from the respiratory drug development and quality control perspective. Each method has unique advantages and disadvantages that influence method selection and data interpretation. Although in vitro methods are used during drug development, they augment rather than substitute in vivo methods.

Characterization of Calu-3 cell monolayers as a model of bronchial epithelial transport: organic cation interaction studies

Background: To fully exploit organic cation transporters for targeted drug delivery in the lung, the use of a readily available and well-characterized tissue culture model and cheap easily detectable substrates is indispensable. Objectives: To investigate the suitability of Calu-3 as tissue model for characterizing organic cation permeation across the bronchial cells using a fluorescent dye, 4-(4-(Dimethylamino)styryl)-N-methylpyridinium iodide (4-DI-1-ASP). Methods: Substrate uptake, inhibition, and transport were performed to establish active transport mechanism. Organic cation transporter expression was determined with quantitative polymerase chain reaction (qPCR), immune-histochemistry, and fluorescent microscopy. Results: 4-Di-1-ASP uptake in Calu-3 cells was concentration (Km = 2.7 ± 0.3 mM, Vmax = 4.6 ± 2.6 nmol/µg protein/30 min), temperature (uptake at 37°C>>4°C), and pH dependent (higher uptake at pH ≥ 7). L-carnitine, verapamil, and corticosterone significantly inhibited its uptake with IC50 of 28.2, 0.81, and 0.12 mM, respectively. Transport of the dye across the cells was polarized (AP→BL transport was 2.5-fold > BL→AP), saturable (Km = 43.9 ± 3.2) (µM; Vmax =0.0228± nmol/cm2/sec) and reduced 3-fold by metabolic inhibition. The expression pattern of the organic cation transporters (OCT) and carnitine/organic cation transporter (OCTN) isoforms was: OCT1<<OCT3 <OCTN1<OCTN2; OCT2 was not detected. Conclusions: Based on qPCR, immunohistochemistry, uptake and transport data, the Calu-3 cells can be used as a model for not only studying strategies for optimizing the effect of inhaled organic cations, but also for cross-validating newly-developed respiratory cell lines.

Differential expression of organic cation transporters in normal and polyps human nasal epithelium: Implications for in vitro drug delivery studies

The aim of this study was to compare the expression of organic cation transporters (OCTs) in normal and polyps nasal epithelium. Primary cell cultures of human nasal epithelium (polyps and normal tissues) were compared by investigating the uptake of a fluorescent organic cation, [4-dimethylaminostyryl-N-methylpyridinium (4-Di-1-ASP)]. The effect of concentration, temperature, pH and competing inhibitors were investigated. Quantitative polymerase chain reaction (qPCR) was used to compare the OCTs gene expression levels in the cells. The K(m) (μM) and V(max) (μM/mg protein/15 min) for 4-Di-1-ASP uptake were higher in normal (K(m)=3031 ± 559.6, V(max)=70.8 ± 8.8) cells compared to polyps (K(m)=952.4 ± 207.8, V(max)=30.9 ± 2.1). qPCR results showed that OCT1-3 and organic cation/carnitine transporter 1-2 gene transcripts (OCTN1-2) were expressed in both normal and polyps cells at comparable levels, with OCT-3 having the highest expression level in both cultures. Kruskal-Wallis ANOVA showed that pH and specific inhibitors had similar effects on both normal and polyps cells (p>0.5). Similarly, OCTs and OCTNs gene expression levels were similar. This study showed that polyps biopsies can be used for isolating cells to study organic cation transporters in human nasal epithelium as no major functional or molecular differences relative to normal cells could be found.

Permeation of losartan across human respiratory epithelium: An in vitro study with Calu-3 cells

Permeation of losartan across human respiratory epithelium: An in vitro study with Calu-3 cells The potential for nasal delivery of losartan, a drug with poor oral bioavailability, was investigated using Calu-3 cells. Epithelial permeation of the drug with or without dimethyl-β-cyclodextrin (DM-β-CD) and glycocholate was investigated. Possible transport mechanism of the compound and epithelial mucosal tolerance were screened. Reversibility of epithelial membrane perturbation was also investigated by measuring transepithelial electrical resistance (TEER) recovery over a 24-h period following drug formulation exposure. The permeability coefficient of losartan was 1.3 ± 0.5 × 10-6 cm s-1. This flux was not significantly different from that of formulations containing DM-β-CD (0.5 and 1.0%) or glycocholate (0.5%). However, the formulation with 1.0% glycocholate significantly increased losartan permeation 7-fold. Losartan flux across the cells was concentration-dependent. Serosal to mucosal permeation was significantly higher than mucosal to serosal permeation. Concentration-dependency, as well as polarity in transport indicated that the flux of the compound across Calu-3 cells was not limited to passive diffusion. Cells exposed to DM-&bT-CD (0.5 and 1.0%) and glycocholate (0.5%) caused no significant change in TEER and mitochondrial dehydrogenase activity (MDH). The results of the study showed that losartan may be a suitable drug candidate for nasal delivery. Permeacija losartana kroz humani respiratorni epitel: In vitro ispitivanja na Calu-3 stanicama U radu je ispitivana mogućnost nazalne primjene losartana, lijeka sa slabom bioraspoloživošću nakon peroralne uporabe, koristeći Calu-3 stanice. Ispitivana je permeacija lijeka kroz epitel u prisutnosti dimetil-β-ciklodekstrina (DM-β-CD) i glikokolata te bez njihove prisutnosti. Predložen je mogući mehanizam transporta kroz epitel i određena je tolerancija epitelne mukoze. Reverzibilnost promjena u epitelu praćena je mjerenjem povrata transepitelnog električnog otpora (TEER) kroz razdoblje od 24 h nakon izlaganju pripravku lijeka. Koeficijent permeabilnosti losartana bio je 1.3 ± 0.46 × 10-6 cm s-1. Taj se dotok značajno ne razlikuje od pripravaka koji sadrže DM-β-CD (0,5 i 1,0 %), odnosno glikokolat (0,5 %) (faktor povećanja ≈ 1,0). Međutim, iz pripravka s 1,0 % glikokolata povećala se permeacija losartana 7 puta. Protok losartana kroz stanice ovisio je o koncentraciji. Permeacija iz seruma u mukozu bila je značajno veća nego u obrnutom smjeru. Ovisnost o koncentraciji te polarnost u transportu ukazuju na to da protok losartana kroz Calu-3 stanice nije ograničen samo na pasivnu difuziju. Stanice izložene dimetil-β-ciklodekstrinu (0,5 i 1,0 %) i glikokolatu (0,5 %) nisu uzrokovale značajne promjene TEER-a i aktivnosti mitohondrijske dehidrogenaze (MDH). Rezultati pokazuju da je losartan pogodan za nazalnu isporuku.

Optimization of human nasal epithelium primary culture conditions for optimal proton oligopeptide and organic cation transporters expression in vitro

AIM To investigate the effect of key tissue culture conditions on cell growth, gene expression and functional uptake of peptide and organic cation transporter substrates in the human nasal epithelium (HNE). METHODS HNE were cultured on different growth surfaces (polystyrene plastic, collagen film, and hydrated collagen gel) and were maintained with three popular nasal tissue culture media supplements [DMEM/F12 supplemented with Ultroser(®) G (2%), FBS (10%) and NuSerum(®) (10%)], respectively. The expression of gene transcripts for organic cation and peptide transporters were screened using qPCR and substrate uptake studies. RESULTS Cell growth surface (polystyrene plastic surface, dried collagen film and hydrated collagen gel) did not significantly alter gene expression levels. However, Ultroser(®) G and FBS caused significant increase in PEPT1, PEPT2, PHT1, OCT3, and OCTN1 levels (~/=2-5-fold for FBS and 2-8-fold for Ultroser(®) G). In terms of the degree to which the supplements affected gene expression, the following observations were made: effect on OCTN1>PEPT2>OCT3>PHT1>PEPT1. Functional uptake of organic cation (4-Di-1-ASP) and peptide [β-Ala-Lys (AMCA)] transporter substrates was significantly lower in cells cultured with NuSerum(®) compared to Ultroser(®) G and FBS cultured cells (p>0.05). CONCLUSIONS Tissue culture media had a major effect on SLC gene expression levels of the human nasal epithelium in primary culture. Ultroser(®) G was identified as the most efficient culture supplement in maintaining SLC transporter expression under most culture conditions, whereas FBS appears to be an economical choice. We do not recommend the use of NuSerum(®) as a supplement for growing HNE for transport studies involving SLC transporters.

Water-soluble organic solubilizers for in vitro drug delivery studies with respiratory epithelial cells: Selection based on various toxicity indicators

The aim of this study was to use different toxicity indices to investigate the effect of N, N-Dimethylacetamide (DMA), Polyethylene glycol 400 (PEG 400), Methyl-Pyrrolidone/aromatic hydrocarbon (MPH), Cremophor® EL, and Dimethylsulfoxide (DMSO) on Calu-3 cells. Membrane perturbation and cytotoxicity were investigated using lactate dehydrogenase (LDH), 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolinium bromide (MTT), transepithelial electrical resistance (TEER) assessment, sodium fluorescein (SF) permeation, and phalloidin actin staining. The MDH activity of cells treated with DMSO (≤ 4.0 v/v), Cremophor EL (≤ 10% v/v), and PEG 400 (≤ 10% v/v) was not significantly altered (p > 0.5). Similarly, DMSO (≤ 8.0% v/v), Cremophor® EL (≤ 16.0% v/v) and PEG 400 (≤ 32.0% v/v) had no significant effect on LDH (p > 0.05). Conversely, MPH and DMA at very low concentrations (≤ 0.25% v/v) induced cell toxicity. However, up to 2.0% v/v MPH and (DMA) had no effect on TEER and SF permeation. No obvious change in actin staining was observed for DMSO (15%), Cremophor® EL (15%), and MPA (1%). However, the actin architecture for cells treated with DMA (1%) and PEG 400 (15%) appeared significantly different from control. Based on this study Cremophor (≤ 10%), PEG 400 (≤ 5%), and DMSO (≤ 5%), and low concentrations of DMA and MPA (≤ 0.25%) were suitable for in vitro studies with Calu-3 cells.

Cytoprotective potential of anti-ischemic drugs against chemotherapy-induced cardiotoxicity in H9c2 myoblast cell line

Abstract To investigate potential prevention or attenuation of anti- cancer drug induced cardiotoxicity using anti-ischemic drugs, a rat myoblast (H9c2) cell line was used as our in vitro cardiac model. Irinotecan and doxorubicin were found to be cytotoxic for the H9c2 cell line with IC50 of 30.69 ± 6.20 and 20.94 ± 6.05 mmol L-1, respectively. 5-Flurouracil and cladribine were not cytotoxic and thus IC50 could not be calculated. When 100 mmol L-1 doxorubicin was incubated for 72 hours with 50 mmol L-1 diltiazem, 100 mmol L-1 dexrazoxane and 100 mmol L-1 losartan, respectively, there was a 58.7 ± 10.2, 52.2 ± 11.7 and 44.7 ± 5.4 % reduction in cell death. When 200 mmol L-1 irinotecan was incubated for 72 hours with 100 mmol L-1 dexrazoxane, losartan and diltiazem, respectively, a 27.7 ± 6.9, 25.6 ± 5.1, and 19.1 ± 2.3 % reduction in cell death was observed. Our data suggests that losartan and diltiazem were as effective as dexrazoxane in protecting the cells against irinotecan- and doxorubicin-induced cell toxicity. These findings offer potential uses of anti- -ischemic drugs for ablation of cytotoxicity in response to mitochondrial injury, thereby improving patient outcomes and reducing health-care costs.

Models for the study of nasal and sinus physiology in health and disease: A review of the literature

Objective Chronic sinusitis is a very common yet poorly understood medical condition with significant morbidity. Hence, it remains an entity that is difficult to treat with unsatisfactory outcomes of current management options. This necessitates research into the etiology and pathophysiology of the condition to enhance our knowledge and the therapeutic options. Unfortunately, this kind of research is not always feasible on human subjects due to practical and ethical limitations. Therefore, an alternative model that simulates the disease had to be found in order to overcome these limitations. These models could either be in vivo or in vitro. The aim of our review is to summarize the research findings and key discoveries of both in vivo and in vitro models of chronic sinusitis that have enhanced our understanding of the condition today and have paved the way for the future research of tomorrow. Data Sources: PubMed literature review. Methods A review of the literature was conducted to identify the main successful in vivo and in vitro models for chronic sinusitis. Results Creating a successful model for chronic sinusitis is no easy task. Over the years, both in vivo animal models and in vitro tissue culture models were proposed, with each model having its accolades and pitfalls, with the ideal model remaining elusive to this day. However, advancing three-dimensional cell culturing techniques seems to be a promising new way to find a more accurate model. Conclusion None of the current models is perfect for a thorough study of chronic sinusitis. However, three-dimensional cell cultures have the potential to bridge the gap between in vivo and in vitro studies. Level of Evidence N/AChronic sinusitis is a very common yet poorly understood medical condition with significant morbidity. Hence, it remains an entity that is difficult to treat with unsatisfactory outcomes of current management options. This necessitates research into the etiology and pathophysiology of the condition to enhance our knowledge and the therapeutic options. Unfortunately, this kind of research is not always feasible on human subjects due to practical and ethical limitations. Therefore, an alternative model that simulates the disease had to be found in order to overcome these limitations. These models could either be in vivo or in vitro. The aim of our review is to summarize the research findings and key discoveries of both in vivo and in vitro models of chronic sinusitis that have enhanced our understanding of the condition today and have paved the way for the future research of tomorrow.

Thyroid hormone (levothyroxine) replacement via the respiratory route by inhalation: in vitro exploratory studies

ABSTRACT Objectives: To conduct proof of principle studies that will enable development of noninvasive (respiratory) delivery systems for levothyroxine (T4). Methods: Preformulation (solubility, stability), formulation and biopharmaceutical (in vitro absorption, transport, gene expression) studies were conducted. Calu-3 cell line was used for permeation studies. Results: Solubility profiles of T4 were established in aqueous (PBS, HBSS, isotonic saline) and non-aqueous solvents (PEG 400, PEG 600, propylene glycol, glycerine). Transport of the compound across Calu-3 cells suggested involvement of active transport systems. This correlated with expression of thyroxine transporters (MCT8, MCT10, OATP1A2, LAT1 and LAT2) in the cell line. Diffusion characteristics showed significant absorption with no detection of T4 metabolite (triiodothyronine). Formulation studies revealed that stable formulations could be prepared using a combination of aqueous and non-aqueous solvents. Conclusions: Results of the studies indicated that T4 can be absorbed effectively from the respiratory mucosa. Factors affecting stability such as pH and temperature should be taken into account during formulation development of this compound for the respiratory route.