The Impact of Inadequate Temperature Storage Conditions on Aggregate and Particle Formation in Drugs Containing Tumor Necrosis Factor-Alpha Inhibitors.

PURPOSE: To measure aggregate and particle formation in tumor necrosis factor-alpha (TNF-α) inhibitors etanercept, adalimumab and certolizumab pegol product samples after exposure to freezing temperature conditions similar to storage conditions previously observed in patients' homes. METHODS: TNF-α inhibitors in their original primary and secondary packaging were exposed to 32 freeze-thaw cycles (-10°C for 120min/5°C for 60 min) or continuous low storage temperature (-20°C for 96 h) before thawing at 2-8°C. Non-stressed products were used as controls. The products were analyzed by high pressure size exclusion chromatography (HP-SEC), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), micro-flow imaging (MFI) and second derivative ultraviolet (UV) spectroscopy. RESULTS: Ten out of twenty-one stressed product samples (47.6%) showed increased particle numbers in the submicron and micron size range when compared to controls. For each product, DLS, MFI and NTA detected an increase in particle level in at least one stressed syringe (both continuous freezing and freeze-thaw), whereas HP-SEC and UV spectroscopy showed no differences between stressed and non-stressed products. CONCLUSION: TNF-α inhibitors are relatively resistant to freezing temperatures similar to storage conditions previously observed in patients' homes. However, almost half of the stressed product samples showed formation of particles in the submicron and micron size range.

Diphtheria toxoid dissolving microneedle vaccination: Adjuvant screening and effect of repeated-fractional dose administration.

In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20 min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5 h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.

Immune modulation by adjuvants combined with diphtheria toxoid administered topically in BALB/c mice after microneedle array pretreatment.

PURPOSE: In this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated. METHODS: TCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers. RESULTS: TCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DT-alum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction. CONCLUSIONS: The potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants.

Nasal administration of an ACTH(4-9) peptide analogue with dimethyl-beta-cyclodextrin as an absorption enhancer: pharmacokinetics and dynamics.

1. The systemic absorption and the neurotrophic effect of the metabolically stabilized ACTH (4-9) analogue, Org2766, were investigated following intranasal (i.n.) administration. 2. Without additives the nasal bioavailability of the peptide was in the order of 15 and 10% in rats and rabbits, respectively. The absorption could be improved by addition of a variety of absorption enhancers to the nasal preparation. The beta-cyclodextrin derivative, dimethyl-beta-cyclodextrin (DM beta CD) at a concentration of 5% (w/v) improved the absorption in rats about 5 fold from 13 +/- 4% (mean +/- s.d.) for administration of the peptide alone to 65 +/- 21%, and in rabbits 1 to 2 fold, from 10 +/- 6% to 17 +/- 8%. 3. The increased permeability of the rat nasal mucosa for Org2766 caused by DM beta CD in rats reversed substantially within 1 h. However, the nasal absorption had not yet completely returned to the level without enhancer. 4. S.c. administered Org2766 accelerated the functional recovery from peripheral nerve damage in rats. However, the peptide did not facilitate nerve repair following i.n. administration with DM beta CD, in spite of the fact that Org2766 was well absorbed. I.v. injection of Org2766 was also ineffective.

Nasal absorption of dihydroergotamine from liquid and powder formulations in rabbits.

Nasal drug delivery is an interesting route of administration for dihydroergotamine in migraine therapy. The currently available formulation contains dihydroergotamine at 4 mg/mL. For a nasal dose of 2 mg, a volume of 0.5 mL has to be administered, which sometimes leads to spillage of the formulation. The aim of the present study was to develop a nasal spray with a dihydroergotamine concentration of 10 mg/mL. To increase the solubility and stability of dihydroergotamine, randomly methylated beta-cyclodextrin was used. Liquid formulations and lyophilized powders of dihydroergotamine and randomly methylated beta-cyclodextrin were prepared. The liquid and powder formulations were compared by determining their pharmacokinetics and absolute bioavailability after nasal administration in rabbits. Nasal sprays were significantly more effective than drops in increasing the nasal bioavailability of dihydroergotamine, but the amount of randomly methylated beta-cyclodextrin in liquid sprays did not significantly alter the nasal absorption. For powder formulations, the dihydroergotamine absorption was dependent on the amount of methylated beta-cyclodextrin and powder volume, and the nasal bioavailability from the optimal powder was slightly, but not significantly, higher than that for liquids. In conclusion, the formulations investigated are a substantial improvement of the current commercial formulation, not only because the spray volume of the liquid spray can be reduced 2.5 times, but also because of the increased stability of liquid and powder sprays with randomly methylated-beta-cyclodextrin.

Classification of cilio-inhibiting effects of nasal drugs.

OBJECTIVE/HYPOTHESIS: Nasal drug formulations are widely used for a local therapeutic effect, but are also used for systemic drug delivery. In the development of new nasal drugs, the toxic effects on the mucociliary clearance and therefore on the ciliated tissue is of importance. In this study, the effect of nasal drugs and their excipients on the ciliary beat frequency (CBF) is investigated. STUDY DESIGN: Experimental, in vitro. METHODS: CBF is measured by a photograph-electric registration method. Excised ciliated chicken trachea tissue is incubated for 15 minutes in the formulation, followed by a reversibility test. To estimate the ciliostatic potential, a classification is given of all tested formulations. According to the CBF, after 60 minutes every drug or excipient could be classified as follows: cilio-friendly: after 60 minutes the CBF has regained 75% or more of its initial frequency; cilio-inhibiting: after 60 minutes the CBF has regained between 25% and 75% of its initial frequency; or ciliostatic: after 60 minutes the CBF has regained 25% or less of its initial frequency. RESULTS: Most formulations used are cilio-friendly or cilio-inhibiting. Only some are ciliostatic. Preservatives have a major role in the cilio-inhibiting effect of the drug. Also, other additives can contribute to the toxicity profile of nasal drug formulations. CONCLUSION: This classification of the cilio-inhibiting potential of nasal drug formulations is a valuable tool in the design of safe nasal drugs. The number of animal studies in vivo can be reduced substantially by using this in vitro screening technique. This study demonstrates that the effect on ciliary movement of most drug formulations is due to the preservatives and/or additives and mostly not to the drug itself.

Effects of N-trimethyl chitosan chloride, a novel absorption enhancer, on caco-2 intestinal epithelia and the ciliary beat frequency of chicken embryo trachea.

N-trimethyl chitosan (TMC) polymers are quaternized chitosans in different degrees of trimethylation. These polymers enhance the absorption of macromolecules through mucosal epithelia by triggering the reversible opening of tight junctions and only allow for paracellular transport. To investigate the safety of these novel absorption enhancers cytotoxicity and ciliotoxicity studies have been performed. Intestinal Caco-2 cell monolayers were chosen to study possible membrane damaging effects of these polymers, using confocal laser scanning microscopy visualization of nuclear staining by a membrane impermeable fluorescent probe during transport of the paracellular marker Texas red dextran (MW 10 000). Ciliated chicken embryo trachea tissue was used to study the effect of the polymers on the ciliary beat frequency (CBF) in vitro. In both studies the TMC polymers of different degrees of substitution (20, 40 and 60%) were tested at a concentration of 1.0% (w/v). No substantial cell membrane damage could be detected on the Caco-2 cells treated with TMCs, while the effect on the CBF in vitro was found to be marginal. TMC60 and TMC40 enhance paracellular transport of Texas red dextran in Caco-2 cell monolayers, whereas TMC20 is ineffective. In conclusion, TMCs of high degrees of substitution may be effective and safe absorption enhancers for peptide and protein drug delivery.

Validation of animal experiments on ciliary function in vitro. I. The influence of substances used clinically.

In vitro studies of ciliary activity require specimens of healthy epithelium in relatively large quantities. Since human material is difficult to obtain, fresh chicken trachea samples have frequently been used in function experiments. The aim of the present study was to investigate whether several substances had comparable effects on the ciliary beat frequency (CBF) of chicken trachea and cryopreserved human respiratory epithelium obtained from the sphenoidal sinus. For this study, we used two topical anaesthetics: cocaine (3% and 7%) and lidocaine (2%). These anaesthetic substances were adjusted to pH 6 and pH 7. We also used two decongestants, namely xylometazoline 0.1% and oxymetazoline 0.1%, and the beta-blocking agent propranolol. Topical anaesthetics appeared to be more ciliostatic in solutions with pH 7 compared to pH 6. Complete ciliostatic effects were reversible, with the exception of the ciliostasis induced by propranolol. The effects of these substances on the CBF of fresh chicken trachea and cryopreserved human tissue did not differ significantly. These experiments show that chicken trachea constitutes a valid substitute for human material in studying ciliary activity in vitro. Moreover, the experiments provide evidence in support of the assumption that cryopreservation has no effect on ciliary reactivity as expressed by the CBF.

Validation of animal experiments on ciliary function in vitro. II. The influence of absorption enhancers, preservatives and physiologic saline.

Ciliary beat frequency (CBF) is one of the most important parameters of mucociliary clearance. Previously, we demonstrated that mucosa from chicken embryo trachea is a good substitute for human ciliated epithelium to study the effects on CBF of substances that are used clinically. In this study, we examined the effect on CBF of four excipients for nasal drug formulations: the absorption enhancers methylated beta-cyclodextrin 2% and sodium taurodihydrofusidate 1%, the preservative benzalkonium chloride 0.01%, and physiologic saline. We also examined the effect on CBF of the cryopreservative dimethyl sulfoxide, which is used to protect ciliated epithelium prior to storage in liquid nitrogen. Results obtained with chicken embryo trachea were compared with those of cryopreserved human mucosa taken from the sphenoidal sinus. For all of the substances tested, the effects on CBF of chicken material were comparable to those measured on human material. Benzalkonium chloride had a stronger ciliostatic effect on human tissue. After 60 min, however, the effect of that substance on CBF was similar in both tissues. We conclude that chicken embryo trachea can be used as a substitute for human ciliated mucosa when studying ciliary activity in vitro.

Absorption enhancing effect of cyclodextrins on intranasally administered insulin in rats.

The absorption enhancing effect of alpha-, beta-, and gamma-cyclodextrin (CD), dimethyl-beta-cyclodextrin (DM beta CD), and hydroxypropyl-beta-cyclodextrin (HP beta CD) on intranasally administered insulin was investigated in rats. Coadministration of 5% (w/v) DM beta CD to the insulin solution resulted in a high bioavailability, 108.9 +/- 36.4% (mean +/- SD, n = 6), compared to i.v. administration, and a strong decrease in blood glucose levels, to 25% of their initial values. Coadministration of 5% alpha-CD gave rise to an insulin bioavailability of 27.7 +/- 11.5% (mean +/- SD, n = 6) and a decrease in blood glucose to 50% of its initial value. The rate of insulin absorption and the concomitant hypoglycemic response were delayed for the alpha-CD-containing solution as compared to the DM beta CD preparation. The other CDs, HP beta CD (5%), beta-CD (1.8%), and gamma-CD (5%), did not have significant effects on nasal insulin absorption. DM beta CD at a concentration of 5% (w/v) induces ciliostasis as measured on chicken embryo tracheal tissue in vitro, but this effect is reversible. In conclusion, DM beta CD is a potent enhancer of nasal insulin absorption in rats.

Effects of absorption enhancers on rat nasal epithelium in vivo: release of marker compounds in the nasal cavity.

PURPOSE: The assessment of the effects of nasal absorption enhancers on the rat nasal epithelium and membrane permeability in vivo after a single nasal dose of the enhancers. METHODS: The release of marker compounds (protein, cholesterol and acid phosphatase) from the nasal epithelium was measured using a lavage technique. The nasal membrane permeability was determined after intravenous administration of a systemic tracer (FITC-albumin). RESULTS: The effects of the absorption enhancers could be classified into four categories. The first consisted of HP beta CD (5%), DM beta CD (2%) and RAMEB (2%) and was not different from the control (physiological saline). For the second category, DM beta CD (5%), effects were significantly higher than for the control. The third category, SGC (1%), was more active than DM beta CD (5%) but less active than the last group. The fourth, most membrane damaging, category consisted of STDHF (1%), laureth-9 (1%) and LPC (1%). Administration of these three enhancers also resulted in release of acid phosphatase, indicating that severe membrane damage occurred. The release of cholesterol from nasal epithelium was largely dependent on the cholesterol solubilisation of the absorption enhancers. The amount of cholesterol released by laureth-9 and LPC was the largest. CONCLUSIONS: The results of this in vivo study are in agreement (i.e. similarity in rank order) with morphological and ciliotoxicity studies of nasal absorption enhancers, demonstrating that this in vivo model is a valuable tool to classify nasal absorption enhancers according to their effects on the rat nasal epithelium.

Hypocalcemic effect of salmon calcitonin following single and repeated nasal and intravenous administration in young rabbits.

The effect of the polypeptide salmon calcitonin (sCT) on serum calcium concentrations following intranasal and intravenous administration was studied in young rabbits. A small, hypocalcemic effect was observed after nasal administration of sCT without additives, indicating that the nasal sCT absorption was low. The absorption could be improved by addition of an absorption-enhancing adjuvant to the nasal preparation. The absorption, however, was still far from complete as was apparent from the much stronger effect of intravenously injected sCT. When a number of sCT doses were given during a 10-week period, the hypocalcemic effect per sCT dose in the young rabbits decreased after intravenous and, although less pronounced, after nasal administration. The decreased response to sCT is probably not related to the induction of neutralizing antibodies or desensitization of sCT receptors, but is more likely associated with the age-dependent level of bone activity.

Simplified solid-phase extraction method for determination of dihydroergotamine in rabbit and human serum using high-performance liquid chromatography with fluorescence detection.

A rapid, selective and sensitive method for the determination of dihydroergotamine (DHE) in serum was developed. Dihydroergocristine (DHEC) was used as an internal standard. Human and rabbit serum samples were extracted using commercial solid-phase cyano (CN) columns. Proteins were washed from these columns with pure acetonitrile, resulting in clean extracts. Extracts were subsequently separated by HPLC in an isocratic way, using a reversed-phase C18 analytical column. Fluorometric detection was performed at excitation and emission wavelengths of 277 and 348 nm, respectively. Calibration curves with amounts of DHE ranging from 2 to 32 ng, were linear. The limit of detection found for DHE was 0.2 ng, extracted from 0.5 ml rabbit or from 2.5 ml human serum. The limit of quantification in serum of both species was 0.7 ng. The method has been shown to be suitable for monitoring DHE in serum during pharmacokinetic studies in rabbits.

Nasal insulin delivery with dimethyl-beta-cyclodextrin as an absorption enhancer in rabbits: powder more effective than liquid formulations.

The nasal absorption of insulin using dimethyl-beta-cyclodextrin (DM beta CD) as an absorption enhancer in rabbits was studied. The nasal administration of insulin/DM beta CD liquid formulations did not result in significant changes in serum insulin and blood glucose concentrations. In contrast, previous experiments in rats showed that the addition of DM beta CD to the liquid nasal formulation resulted in an almost-complete insulin absorption, with a concomitant strong hypoglycaemic response. Apparently, the effect of the cyclodextrin derivative on insulin absorption differs between animal species following nasal delivery of insulin/DM beta CD solutions. On the other hand, nasal administration of the lyophilized insulin/DM beta CD powder dosage form in rabbits resulted in increased serum insulin concentrations, and a maximum decrease in blood glucose of about 50%. The absolute bioavailability of the nasally administered insulin/DM beta CD powder was 13 +/- 4%, compared to 1 +/- 1% for both an insulin/DM beta CD liquid and an insulin/lactose powder formulation. It is concluded that insulin powder formulations with DM beta CD as an absorption enhancer are much more effective than liquid formulations.

Methylated beta-cyclodextrins are able to improve the nasal absorption of salmon calcitonin.

The absorption enhancing effect of methylated beta-cyclodextrins on the nasal absorption of salmon calcitonin (sCT) was studied in rats and rabbits. The nasal absorption of sCT following administration without additives was low in both species. The absorption in rats could be largely improved by coadministration of cyclodextrins as apparent from the effect on serum calcium concentrations. Trimethyl-beta-cyclodextrin (TM beta CD), at a concentration of 5% (w/v), was the least potent enhancer. Randomly methylated-beta-cyclodextrin (RM beta CD) and dimethyl-beta-cyclodextrin (DM beta CD), all at a concentration of 5% (w/v), were almost equally effective in decreasing serum calcium levels, and the hypocalcemic responses were similar to those of i.v. and s.c. injected sCT. Absorption enhancement was already achieved with 1% DM beta CD added to the nasal formulations. In rabbits, only the effect of DM beta CD on the nasal sCT absorption was investigated. A total serum calcium decrement in 4 hours of 9.4 +/- 3.9% (mean +/- SD) was observed following nasal administration of 12.6 IU/kg sCT with 5% DM beta CD, comparable to that of i.v.-injected sCT. In conclusion, the methylated cyclodextrins DM beta CD and RM beta CD are suitable absorption enhancers for nasal sCT administration, which is expected to have a clinical impact on the therapy with calcitonin.

Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats: effects of absorption enhancers.

PURPOSE: To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivo using confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated beta-cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. METHODS: Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. RESULTS: Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated beta-cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated beta-cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. CONCLUSIONS: Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.