Remigius Uchenna Agu

The lung as a route for systemic delivery of therapeutic proteins and peptides

The large surface area, good vascularization, immense capacity for solute exchange and ultra-thinness of the alveolar epithelium are unique features of the lung that can facilitate systemic delivery via pulmonary administration of peptides and proteins. Physical and biochemical barriers, lack of optimal dosage forms and delivery devices limit the systemic delivery of biotherapeutic agents by inhalation. Current efforts to overcome these difficulties in order to deliver metabolic hormones (insulin, calcitonin, thyroid-stimulating hormone [TSH], follicle-stimulating hormone [FSH] and growth hormones) systemically, to induce systemic responses (immunoglobulins, cyclosporin A [CsA], recombinant-methionyl human granulocyte colony-stimulating factor [r-huG-CSF], pancreatic islet autoantigen) and to modulate other biological processes via the lung are reviewed. Safety aspects of pulmonary peptide and protein administration are also discussed.

In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studies

The aim of this study was to establish a collagen matrix‐based nasal primary culture system for drug delivery studies. Nasal epithelial cells were cultured on derivatised (Cellagen membrane CD‐24), polymerised (Vitrogen gel) and fibrillar (Vitrogen film) collagen substrata. Cell morphology was assessed by microscopy. The cells were further characterised by measurement of ciliary beat frequency (CBF), transepithelial resistance (TER), permeation of sodium fluorescein, mitochondrial dehydrogenase (MDH) activity and lactate dehydrogenase (LDH) release upon cell exposure to sodium tauro‐24, 25 dihydrofusidate (STDHF). Among the three collagen substrata investigated, the best epithelial differentiated phenotype (monolayer with columnar/cuboidal morphology) occurred in cells grown on Cellagen membrane CD‐24 between day 4 and day 11. Cell culture reproducibility was better with Cellagen membrane CD‐24 (90%) in comparison with Vitrogen gel (70%) and Vitrogen film (< 10%). TER was higher in cells grown on Vitrogen gel than on Cellagen membrane CD‐24 and Vitrogen film. The apparent permeability coefficient (Papp × 10−7 cm s−1) of sodium fluorescein in these conditions was 0.45 ± 0.08 (Vitrogen gel) and 1.91 ± 0.00 (Cellagen membrane CD‐24). Except for LDH release, CBF and cell viability were comparable for all the substrata. Based on MDH activity, LDH release, CBF, TER and permeation studies, Cellagen membrane CD‐24‐ and Vitrogen gel‐based cells were concluded to be functionally suitable for in‐vitro nasal drug studies. Vitrogen film‐based cultures may be limited to metabolism and cilio‐toxicity studies.

Scintigraphic evaluation in rabbits of nasal drug delivery systems based on carbopol 971p® and carboxymethylcellulose

The residence time of apomorphine mucoadhesive preparations incorporating 99mTc labeled colloidal albumin in rabbit nasal cavity was evaluated by gamma scintigraphy. This technique was used to compare the nasal clearance of preparations based either on Carbopol 971P((R)) or lactose (control), each with and without apomorphine, or carboxymethylcellulose with apomorphine. The planar 1-min images showed an excipient-dependent progressive migration of radioactivity with time from the nasal cavity to the stomach and intestine. Thirty minutes post insufflation, the percentages of the formulations cleared from the nasal cavity were 47% for lactose, 26% for lactose/apomorphine, 10% for Carbopol 971P((R)), and 3% for both Carbopol 971P((R))/apomorphine and carboxymethylcellulose/apomorphine. Three hours post insufflation, the percentages of the formulations cleared from the nasal cavity were 70% for lactose, 58% for lactose/apomorphine, 24% for Carbopol 971P((R)), 12% for Carbopol 971P((R))/apomorphine, and 27% for carboxymethylcellulose/apomorphine. Apomorphine inhibited nasal mucociliary clearance since migration of the radioactivity administered with apomorphine containing preparations was in all cases slower than that of the corresponding powder without apomorphine. The peak plasma concentration of apomorphine was attained while all the formulations were still within the nasal cavity. The use of mucoadhesive polymers such as Carbopol 971P((R)) or carboxymethylcellulose in nasal dosage forms increases their residence time within the nasal cavity and provides the opportunity for sustained nasal drug delivery.

Nasal toxicological investigations of Carbopol 971P formulation of apomorphine: effects on ciliary beat frequency of human nasal primary cell culture and in vivo on rabbit nasal mucosa.

OBJECTIVE The objective of this study was to investigate the nasal toxicity of a mucoadhesive Carbopol 971P formulation of apomorphine. MATERIALS AND METHODS The effects of different concentrations of Carbopol 971P and apomorphine on ciliary beat frequency (CBF) were studied in suspension cultures of human nasal epithelial cells. The rabbit nasal mucosal tolerance of the formulation and its components were investigated using light microscopy. Different groups of the rabbits received twice daily, air puffs, glucose, glucose/apomorphine, Carbopol 971P or Carbopol 971P/apomorphine for 1 week (glucose-treated rabbits) or 1, 2 and 4 weeks (other treatments). RESULTS Both Carbopol 971P and apomorphine showed both concentration- and time-dependent inhibitory effects on the CBF. The effects on CBF were: apomorphine, 1.0% w/v, irreversible ciliostasis; 0.1 and 0.5% w/v, reversible cilio-inhibition; 0.01%w/v, irreversible cilio-stimulation; and Carbopol 971P, 0.1 and 0.25% w/v, partially-reversible cilio-inhibition. Glucose and glucose/apomorphine physical mixture caused mild inflammation. Carbopol 971P (both with and without apomorphine) caused severe inflammation, which increased with duration of treatment. Necrosis, squamous metaplasia or ciliary degeneration was not observed. CONCLUSIONS Due to the severe inflammation caused by Carbopol 971P with and without apomorphine, we conclude that this polymer is not a suitable carrier for intranasal administration of apomorphine. This is in spite of the reversible effects of Carbopol 971P (0.1 and 0. 25% w/v) and apomorphine (0.1 and 0.5% w/v) on CBF.

Nasal absorption enhancement strategies for therapeutic peptides: an in vitro study using cultured human nasal epithelium.

This study examined the potential usefulness of cultured human nasal epithelium as a model to investigate nasal absorption enhancement strategies for therapeutic peptides. The transport of leucine enkephalin (Leu-Enk) in the presence of bestatin and puromycin, respectively and various combinations of these protease inhibitors with absorption enhancers capable of inhibiting proteases or protecting peptides against protease degradation (glycocholate, dimethyl-beta-cyclodextrin (DM beta CD)) was studied. Epithelial membrane perturbation, protein leakage, bestatin/puromycin absorption and rebound aminopeptidase activity were used as toxicological end-points. The combination of puromycin with glycocholate or DM beta CD resulted in a higher absorption enhancement of Leu-Enk (9-14%) than when the absorption enhancers were combined with bestatin (1-3%) or when the inhibitors were used alone (2-4%). The higher absorption enhancement resulting from the combination of protease inhibitors with absorption enhancers caused a significant reduction of epithelial resistance and increased sodium fluorescein transport. Although only puromycin permeated the human nasal epithelium, both protease inhibitors induced a significant rebound aminopeptidase activity (25-61%), which can be associated with protein leakage (21-46%). This study highlighted (i) the potential usefulness of cultured human nasal epithelium as a model to study nasal absorption enhancement of therapeutic peptides; (ii) further studies using in vivo nasal models are required to ascertain whether the membrane perturbation and cytotoxicity observed with various combinations of the protease inhibitors and absorption enhancers really raise safety concerns.

Safety assessment of selected cyclodextrins — effect on ciliary activity using a human cell suspension culture model exhibiting in vitro ciliogenesis

The objective of this study was to assess the cilio-inhibitory effect of a series of cyclodextrins using a human cell suspension culture system exhibiting in vitro ciliogenesis. Enzymatically released human nasal epithelial cells were cultured as sequential monolayer-suspension culture showing in vitro ciliogenesis. Ciliary beat frequency (CBF) was determined by computerized microscope photometry. Among the cyclodextrins investigated (gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, anionic-beta-cyclodextrin polymer, dimethyl-beta-cyclodextrin and alpha-cyclodextrin), it was shown that after 30 min of exposure, gamma-cyclodextrin (10% w/v), hydroxypropyl-beta-cyclodextrin (10.0% w/v) and anionic-beta-CD polymer (8.0% w/v) were not significantly cilio-inhibitory (P0.05). Similarly, CBF remained stable upon cell exposure to alpha-cyclodextrin (2.0% w/v) and dimethyl-beta-cyclodextrin (1.0% w/v). However, higher concentrations of alpha-cyclodextrin and dimethyl-beta-cyclodextrin resulted in mild to severe cilio-inhibition after 45 min of exposure. The effect of alpha-cyclodextrin (5.0% w/v; 54+/-4% cilio-inhibition) was partially reversible while dimethyl-beta-cyclodextrin (10% w/v; 36+/-4% cilio-inhibition) was irreversible. The cilio-inhibition observed in this model was lower than reported for chicken trachea model. Given the fact that (1) irreversible cilio-inhibition observed in this study occurred only at concentrations exceeding those used in pharmaceutical formulations and/or at an unusual exposure time (45 min) and that (2) in an in vivo situation, dilution and mucociliary clearance contribute to further decrease in local concentrations of the applied compound, the results of this study confirm the safety of the cyclodextrins investigated as nasal absorption enhancers.

Effects of pharmaceutical compounds on ciliary beating in human nasal epithelial cells: a comparative study of cell culture models

AbstractPurpose. To test two in vitro human nasal epithelial cell culture systems for their ability to screen the effects of pharmaceutical compounds on ciliary beating. Methods. Human nasal epithelial cells were cultured as monolayer and in a sequential monolayer-suspension culture with in vitro ciliogenesis. The influence of reference cilio-stimulatory compounds (glycocholate, isoprenaline), reference cilio-inhibitory compounds (chlorocresol, diphenhydramine) and pH on ciliary beating was investigated using computerized microscope photometry. Results. Sodium glycocholate (0.5% w/v) maximally and reversibly increased CBF of the cells in both culture systems by 26 ± 4% (monolayer) and 18 ± 6% (suspension). Similarly, isoprenaline (10-3 M) maximally, but irreversibly increased CBF of the cells by 14 ± 3% (monolayer) and 17 ± 4% (suspension). Chlorocresol (0.005% w/ v) reversibly reduced the CBF of the cells by 50 ± 6% (monolayer) and 34 ± 4% (suspension); at a higher concentration (0.1% w/v) it resulted in instantaneous and irreversible ciliostasis. Diphenhydramine (0.1% w/v) reversibly reduced CBF in both culture systems by 45 ± 13% (monolayer) and 69 ± 5% (suspension); irreversible cilio-stasis occurred in less than 2 minutes in both culture systems upon cell exposure to diphenhydramine (1.0% w/v). In the monolayer culture system, CBF was stable only within the physiological pH range of 6.5−8.0; ciliary beating in the suspension culture remained stable within a pH range of 4.0−10.0. Conclusions. Both cell culture systems are suitable for screening the effects of pharmaceutical compounds on ciliary beating. Especially the sequential monolayer-suspension culture appears to be very promising as ciliary activity can be preserved for as long as 6 months.

Toxicological investigations of the effects carboxymethylcellulose on ciliary beat frequency of human nasal epithelial cells in primary suspension culture and in vivo on rabbit nasal mucosa

The objective of this study was to investigate the safety of a mucoadhesive carboxymethylcellulose (CMC) formulation for intranasal administration of apomorphine. The effect of different concentrations of CMC on ciliary beat frequency (CBF) was studied using a human nasal epithelial suspension cell culture system. The CBF was determined by computerized microscope photometry. The in vivo rabbit nasal mucosal tolerance of the mucoadhesive polymer was investigated using light microscopy. Twice daily, six rabbits received CMC powder in one nostril and CMC/apomorphine powder in the alternate nostril for 4 weeks. Two control rabbits received air puffs in one nostril and nothing in the alternate nostril. The rabbits were subsequently sacrificed and the stained nasal sections examined microscopically. CMC showed both concentration- and time-dependent inhibitory effects on the CBF. Only mild-to-moderate cilio-inhibition was recorded with the different concentrations of the polymer. CMC (both with and without apomorphine) caused mild-to-moderate inflammation after 4 weeks. Necrosis, squamous metaplasia or ciliary degeneration was not observed. Based on: (1) the mild-to-moderate cilio-inhibition induced by different concentrations of CMC; and (2) the mild-to-moderate nasal mucosal inflammation caused by CMC with and without apomorphine, we conclude that this polymer can be considered as a safe carrier for short-term intranasal administration. However, further investigations are required for its use in the treatment of chronic diseases such as with apomorphine in Parkinsons disease.

Metabolism and absorption enhancement of methionine enkephalin in human nasal epithelium

The objective of this study was to investigate absorption enhancing approaches for systemic delivery of methionine enkephalin via the nose. Absorption promotion of methionine enkephalin in the presence of protease inhibitors (bestatin, puromycin) and absorption enhancers (glycocholate, dimethyl-beta-cyclodextrin) were investigated in human nasal epithelium. Co-administration of the peptide with protease inhibitors and absorption enhancers resulted in a remarkable increase in Met-Enk permeation (4- to 94-fold). The increase was proportional to transepithelial resistance reduction and permeation of paracellular marker dye. Perturbation of the epithelial tight junctions seen in vitro may not occur in vivo due to mucus protection and mucociliary clearance.

Proton-Coupled Oligopeptide Transporter (POT) Family Expression in Human Nasal Epithelium and Their Drug Transport Potential

The molecular and functional expression of peptide transporters (PEPT1 and PEPT2, PHT1, PHT2) in human nasal epithelium was investigated. Quantitative/reverse transcriptase polymerase chain reaction (qPCR/RT-PCR), Western blotting and indirect immuno-histochemistry were used to investigate the functional gene and protein expression for the transporters. Uptake and transport studies were performed using metabolically stable peptides [β-alanyl-L-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA) and β-alanyl-L-histidine (carnosine)]. The effects of concentration, temperature, polarity, competing peptides, and inhibitors on peptide uptake and transport were investigated. PCR products corresponding to PEPT1 (150 bp), PEPT2 (127 bp), PHT1 (110 bp) and PHT2 (198 bp) were detected. Immunohistochemistry and Western blotting confirmed the functional expression of PEPT1 and PEPT2 genes. The uptake of β-Ala-Lys-AMCA was concentration-dependent and saturable (Vmax =4.1 ( 0.07 μmol/min/mg protein, Km = 0.6 ( 0.07 μM). The optimal pH for intracellular accumulation of β-Ala-Lys-AMCA was 6.5. Whereas dipeptides and carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited peptide uptake and transport, L-Phe had no effect on peptide transport. The permeation of β-alanyl-L-histidine was concentration-, direction-, and temperature-dependent. The uptake, permeation, qPCR/RT-PCR and protein expression data showed that the human nasal epithelium functionally expresses proton-coupled oligopeptide transporters.