Biosimilar rhG-CSFs: how similar are they?

Biosimilars are more affordable copycat versions of originator biological products in much the same way as generics are copies of small molecule pharmaceuticals. However, while generics are approved on the basis of structural and pharmacokinetic equivalence, the intricate structure of biological medicinal products and the complex nature of their manufacturing process in living organisms impose a separate, and more stringent, regulatory approval process. The aims of this article are (a) to discuss key aspects of the development process and authorization requirements for biosimilars in Europe using published comparative physicochemical and clinical data for EU-approved recombinant human granulocyte colony stimulating factor (rhG-CSF) biosimilars, the most recent addition to the therapeutic category of hematopoietic growth factors, and (b) to demonstrate that the rigorous scientific evaluation process is designed to ensure that their clinical safety and efficacy is not compromised by the abbreviated development program.

Effect of propylene glycol on ibuprofen absorption into human skin in vivo.

The objective was to assess the impact of propylene glycol (PG), a common cosolvent in topical formulations, on the penetration of ibuprofen into human skin in vivo. Drug uptake into the stratum corneum (SC), following application of saturated formulations containing from 0 to 100% v/v PG, was assessed by tape-stripping. Dermatopharmacokinetic parameters, characterizing drug amount in and diffusivity through the SC, were derived. The solubility behavior of ibuprofen in PG-water mixtures was carefully evaluated, as were a number of other physical properties. Ibuprofen delivery depended on the level of PG in the vehicle, despite all formulations containing the drug at equal thermodynamic activity. PG appeared to alter the solubility of ibuprofen in the SC (presumably via its own uptake into the membrane), the effect becoming more important as the volume fraction of cosolvent in the formulation increased. In summary, tape-stripping experiments, with careful interpretation, can reveal details of a drug's bioavailability in the skin following topical application and may be used to probe the mechanism(s) by which certain excipients influence local drug delivery.

In vivo methods for the assessment of topical drug bioavailability.

This paper reviews some current methods for the in vivo assessment of local cutaneous bioavailability in humans after topical drug application. After an introduction discussing the importance of local drug bioavailability assessment and the limitations of model-based predictions, the focus turns to the relevance of experimental studies. The available techniques are then reviewed in detail, with particular emphasis on the tape stripping and microdialysis methodologies. Other less developed techniques, including the skin biopsy, suction blister, follicle removal and confocal Raman spectroscopy techniques are also described.

Ibuprofen transport into and through skin from topical formulations: in vitro-in vivo comparison.

The goal was to compare ibuprofen transport into and through skin in vivo in man and in vitro (across silicone membranes and freshly excised pig skin) from four marketed formulations. Ibuprofen gels were administered in vivo for 30 minutes. The stratum corneum (SC) at the application site was then tape-stripped, quantified gravimetrically, and extracted for drug analysis. Together with concomitant transepidermal water loss measurements, SC drug concentration-depth profiles were reproducibly determined and fitted mathematically to obtain a partition coefficient, a first-order rate constant related to ibuprofen diffusivity, and the total drug amount in the SC at the end of the application. All derived parameters were consistent across formulations. Ibuprofen permeation data through both silicone membrane and pig ear skin were also fitted to yield partitioning and diffusion parameters. The former revealed that ibuprofen partitioned differently from the gels into this model barrier. Across pig skin, however, better correlation with in vivo results was found. The dermatopharmacokinetic approach, using SC tape-stripping, offers a valid method to assess equivalency between topical drug formulations. In vitro experiments must be extrapolated cautiously to the clinic, especially when complex interactions between real formulations, which deliver both drug and excipients, and the skin occur.

Dermatopharmacokinetic prediction of topical drug bioavailability in vivo.

The overall goal of this study was to explore the potential of using stratum corneum (SC) tape-stripping, post-application of a topical drug formulation, to derive dermatopharmacokinetic parameters describing the rate and extent of delivery into the skin. Ibuprofen was administered in 75:25 v/v propylene glycol-water to the ventral forearms of human volunteers for periods ranging between 15 and 180 minutes. Subsequently, SC was tape-stripped, quantified gravimetrically, and extracted for drug analysis. Together with concomitant transepidermal water loss measurements, SC concentration-depth profiles of the drug were reproducibly determined and fitted mathematically. The SC-vehicle partition coefficient (K) and a first-order rate constant related to ibuprofen diffusivity in the membrane (D/L2, where L=SC thickness) were derived from data-fitting and characterized the extent and rate of drug absorption across the skin. Integration of the concentration profiles yielded the total drug amount in the SC at the end of the application period. Using K and D/L2 obtained from the 30-minute exposure, it was possible to predict ibuprofen uptake as a function of time into the SC. Prediction and experiment agreed satisfactorily suggesting that objective and quantitative information, with which to characterize topical drug bioavailability, can be obtained from this approach.

Structure-permeation relationships for the non-invasive transdermal delivery of cationic peptides by iontophoresis.

Transdermal iontophoresis enables the controlled, non-invasive administration of peptide therapeutics. The aims of this study were (i) to evaluate the effect of amino acid sequence and the spatial distribution of peptide physicochemical properties on electrotransport, and (ii) to develop a quantitative model to predict peptide transport rates. Experimental results showed that the distribution of molecular properties over the peptide surface significantly affected iontophoretic delivery: different arrangements of the same residues resulted in different transport behavior. Computational studies generated three-dimensional quantitative structure-permeation relationships (3D-QSPR) based on 3D descriptors. The model predicted that iontophoresis was favored by peptide hydrophilicity but hindered by voluminous, localized hydrophobicity. Molecular characteristics that favor electrotransport are the converse of those required for passive diffusion across biological membranes. The data represent the first analysis of peptide electrotransport in terms of the spatial distribution of molecular properties and provide insight into the ab initio prediction of transdermal iontophoretic peptide delivery.

Pig ear skin ex vivo as a model for in vivo dermatopharmacokinetic studies in man.

OBJECTIVE: The objective was to investigate pig ear skin as a surrogate for human skin in the assessment of topical drug bioavailability by sequential tape-stripping of the stratum corneum (SC). The potential benefits of ex vivo investigations are manifold: ethical approval is not required, multiple replicate experiments are more easily performed, and toxic compounds can be evaluated. MATERIALS AND METHODS: Ex vivo experiments on isolated pig ears were compared with in vivo studies in human volunteers. Four formulations, comprising the model drug, ibuprofen, in different propylene glycol (PG)-water mixtures (25:75, 50:50, 75:25 and 100:0), were compared. RESULTS: Derived dermatopharmacokinetic parameters characterizing the diffusion and partitioning of the drug in the SC ex vivo were consistent with those in vivo following a 30-minute application period. Further, the non-steady-state ex vivo results could be used to predict the in vivo concentration profile of the drug across the SC when a formulation was administered for 3 h (i.e., close to steady-state). CONCLUSIONS: Taken together, the results obtained suggest that pig ear skin ex vivo has promise as a tool for topical formulation evaluation and optimization.

Topical iontophoresis of valaciclovir hydrochloride improves cutaneous aciclovir delivery.

PURPOSE: To investigate the topical iontophoresis of valaciclovir (VCV) as a means to improve cutaneous aciclovir (ACV) delivery. METHODS: ACV and VCV electrotransport experiments were conducted using excised porcine skin in vitro. RESULTS: While the charged nature of the prodrug, VCV, enabled it to be more efficiently iontophoresed into the skin than the parent molecule, ACV, only the latter was detectable in the receptor chamber, suggesting that VCV was enzymatically cleaved into the active metabolite during skin transit. Iontophoresis of VCV was significantly more efficient than that of ACV; the cumulative permeation of ACV after 1, 2 and 3 h of VCV iontophoresis at 0.5 mA cm(-2) and using an aqueous 2 mM (approximately 0.06%) formulation was 20+/-10, 104+/-47 and 194+/- 82 microg cm( -2), respectively (cf. non-quantifiable levels, 0.1 and 1.0+/-0.7 microg cm(-2) after ACV iontophoresis). CONCLUSIONS: These delivery rates provide ample room to reduce either current density or the duration of current application. Preliminary in vitro data serve to emphasize the potential of VCV iontophoresis to improve the topical therapy of cutaneous herpes simplex infections and merit further investigation to demonstrate clinical efficacy.

Emerging strategies for the transdermal delivery of peptide and protein drugs.

Transdermal delivery has been at the forefront of research addressing the development of non-invasive methods for the systemic administration of peptide and protein therapeutics generated by the biotechnology revolution. Numerous approaches have been suggested for overcoming the skin's formidable barrier function; whereas certain strategies simply act on the drug formulation or transiently increase the skin permeability, others are designed to bypass or even remove the outermost skin layer. This article reviews the technologies currently under investigation, ranging from those in their early-stage of development, such as laser-assisted delivery to others, where feasibility has already been demonstrated, such as microneedle systems, and finally more mature techniques that have already led to commercialisation (e.g., velocity-based technologies). The principles, mechanisms involved, potential applications, limitations and safety considerations are discussed for each approach, and the most advanced devices in each field are described.

Capillary zone electrophoresis for the estimation of transdermal iontophoretic mobility.

The objective of the study was to investigate the relationship between transdermal iontophoretic flux--specifically, the electromigratory component--and electrophoretic mobility as determined by capillary zone electrophoresis (CZE). First, the steady-state iontophoretic transport rates of a series of dipeptides across porcine skin were determined in vitro. Co-iontophoresis of acetaminophen was used to quantify the respective contributions of electroosmosis (EO) and electromigration (EM). Second, the electrophoretic mobilities of the dipeptides and three other cationic drugs (lidocaine, propranolol, and quinine) were determined, under equivalent experimental conditions, using CZE. Analysis of the transport data using the results of the CZE experiments revealed a linear dependence (r2 > 0.9) between EM flux and electrophoretic mobility. The CZE measurements also provided insight into the charge state of "zwitterionic" dipeptides, H-Glu-epsilon-Lys-OH and H-Tyr-Gln-OH, revealing that these molecules had partial net negative charges under the formulation conditions, accounting for the absence of anodal iontophoretic delivery. The results suggest that CZE might (i) enable identification of ionization states of complex molecules, (ii) serve as a preliminary screen to identify electrically mobile compounds suitable for iontophoretic delivery, and (iii) prove useful for predicting the EM contribution to transdermal iontophoretic flux.

Contributions of electromigration and electroosmosis to peptide iontophoresis across intact and impaired skin.

d-(Arg)-Kyotorphin iontophoresis was investigated across intact and impaired skins in vitro. Iontophoretic flux increased from 68+/-12 to 538+/-116 nmol cm(-2) h(-1) when the peptide concentration in the anodal compartment was raised from 5 to 40 mM. Electromigration was the principal transport mechanism, accounting for approximately 70% of total peptide delivery. Reducing the number of competing ions in the formulation significantly increased iontophoretic flux but did not affect convective solvent flow. The latter was independent of peptide concentration indicating that skin permselectivity was not modified by kyotorphin transport. Total iontophoretic flux was unaffected when the stratum corneum was removed by tape-stripping (146+/-34 versus 150+/-26 nmol cm(-2) h(-1)). However, the contributions of the different transport mechanisms were significantly altered: (i) electromigration decreased, as more of the charge was carried by anions from the sub-dermal milieu; (ii) electroosmosis was absent; and (iii) passive permeation increased significantly. Transport rates across intact and impaired skin barriers were statistically indistinguishable when the donor electrolyte composition was modified; increased competition from anions was mitigated by the decreased Na+ levels in the formulation. Removal of Cl- ions from the receiver phase further increased peptide delivery, and also increased anodal electroosmosis.

Effect of charge and molecular weight on transdermal peptide delivery by iontophoresis.

PURPOSE: The study was conducted to investigate the impact of charge and molecular weight (MW) on the iontophoretic delivery of a series of dipeptides. METHODS: Constant current iontophoresis of lysine and 10 variously charged lysine- and tyrosine-containing dipeptides was performed in vitro. RESULTS: Increasing MW was compensated by additional charge; for example, Lys (MW = 147 Da, +1) and H-Lys-Lys-OH (MW = 275 Da, +2) had equivalent steady-state fluxes of 225 +/- 48 and 218 +/- 40 nmol cm(-2) h(-1), respectively. For peptides with similar MW, e.g., H-Tyr-D-Arg-OH (MW = 337 Da, +1) and H-Tyr-D-Arg-NH(2) (MW = 336 Da, +2), the higher valence ion displayed greater flux (150 +/- 26 vs. 237 +/- 35 nmol cm(-2) h(-1)). Hydrolysis of dipeptides with unblocked N-terminal residues, after passage through the stratum corneum, suggested the involvement of aminopeptidases. The iontophoretic flux of zwitterionic dipeptides was less than that of acetaminophen and dependent on pH. CONCLUSIONS: For the series of dipeptides studied, flux is linearly correlated to the charge/MW ratio. Data for zwitterionic peptides indicate that they do not behave as neutral ("charge-less") molecules, but that their iontophoretic transport is dependent on the relative extents of ionization of the constituent ionizable groups, which may also be affected by neighboring amino acids.

Transdermal iontophoretic delivery of triptorelin in vitro.

The feasibility of delivering triptorelin ([D-Trp6]LHRH) by transdermal iontophoresis was evaluated in vitro. Peptide electrotransport at different current densities and donor concentrations was measured across porcine ear skin. The concomitant delivery of an electroosmotic marker enabled calculation of the respective contributions of electromigration (EM) and electroosmosis (EO) to iontophoretic delivery. At a given concentration (3 mM), a threefold increase in current density produced a corresponding increase in the cumulative amount of peptide present in the receptor compartment. Conversely, doubling the concentration to 6 mM produced a twofold reduction in the amount of peptide delivered, partly due to a concentration-dependent inhibition of EO. EM was revealed to be the predominant transport mechanism, accounting for 80% of overall delivery. Finally, despite the inhibition of EO, the results indicate that application of an iontophoretic current of 0.8 mA over a relatively small contact area (4 cm2) would provide a delivery rate of 36 microg/h, largely sufficient for therapeutic requirements.

Effect of amino acid sequence on transdermal iontophoretic peptide delivery.

The objective of this study was to investigate the effect of amino acid sequence on the transdermal delivery of peptides by iontophoresis. Structurally related, cationic tripeptides based on the residues at positions (i) 6-8 in LHRH (Ac-X-Leu-Arg-NH(2)) and (ii) 3-5 in octreotide (Ac-X-dTrp-Lys-NH(2)) were studied. Iontophoretic transport experiments were conducted using porcine skin in vitro to investigate the dependence of flux on peptide concentration. Co-iontophoresis of acetaminophen enabled deconvolution of the contributions of electromigration (EM) and electroosmosis (EO) and the calculation of an electroosmotic inhibition factor (IF). A two-fold increase in donor peptide concentration increased iontophoretic flux for most peptides, and electroosmotic inhibition for dNal-containing tripeptides. The improvement in transport and the impact on the EM and EO components were peptide-specific. A reduction in the number of competing ions in the formulation significantly increased transport and, specifically, the EM contribution; it also increased IF of compounds with a propensity to interact with the membrane. No monotonic dependence of flux on either molecular weight or lipophilicity was observed. Iontophoretic peptide transport could not be rationalized in terms of either peptide molecular weight or computational 2D estimates of lipophilicity. Data suggest that a more complex three-dimensional approach is required to develop structure permeation relationships governing iontophoretic peptide delivery.

Transdermal iontophoretic delivery of vapreotide acetate across porcine skin in vitro.

PURPOSE: The purpose of this study was to evaluate the feasibility of delivering vapreotide, a somatostatin analogue, by transdermal iontophoresis. METHODS: In vitro experiments were conducted using dermatomed porcine ear skin and heat-separated epidermis. In addition to quantifying vapreotide transport into and across the skin, the effect of peptide delivery on skin permselectivity was also measured. The influence of (1) current density, (2) pre- and post-treatment of the skin, (3) competitive ions, and (4) inclusion of albumin in the receptor on vapreotide delivery were investigated. RESULTS: Epidermis proved to be a better model than dermatomed skin for vapreotide transport studies. Despite the susceptibility of vapreotide to enzymatic degradation, a flux of 1.7 microg/cm2 per hour was achieved after 7 h of constant current iontophoresis (0.15 mA/cm2). Post-iontophoretic extraction revealed that, depending on the experimental conditions, 80-300 microg of peptide were bound to the skin. Vapreotide was found to interact with the skin and displayed a current-dependent inhibition of electroosmosis. However, neither the pre-treatment strategies to saturate the putative binding sites nor the post-treatment protocols to displace the bound peptide were effective. CONCLUSION: Based on the observed transport rate of vapreotide across porcine epidermis and its clinical pharmacokinetics, therapeutic concentrations should be achievable using a 15-cm2 patch.

Enhancement of topical delivery from biodegradable nanoparticles.

PURPOSE: To determine whether and how encapsulation of lipophilic compounds in polymeric nanoparticles is able to improve topical delivery to the skin. METHODS: The penetration of octyl methoxycinnamate (OMC; Parsol MCX), a highly lipophilic sunscreen, into and across porcine ear skin in vitro was investigated, subsequent to encapsulation in poly(epsilon-caprolactone) nanoparticles, using tape-stripping. Confocal laser scanning microscopy (CLSM) was used to visualize the distribution of nanoparticles, charged with Nile red (NR), a lipophilic and fluorescent dye. RESULTS: Quantification of OMC in the skin using tape-stripping demonstrated that nanoparticulate encapsulation produced a 3.4-fold increase in the level of OMC within the stratum corneum (SC), although the use of nanoparticles did not appear to increase skin permeation (it was not possible to detect OMC in the receiver compartment after 6 h). The confocal images showed that the fluorescence profile observed in the skin after application of NR-containing nanoparticles was clearly different from that seen following application of NR dissolved in propylene glycol. Two hours postapplication of NR-containing nanoparticles, fluorescence was perceptible at greater depths (up to 60 microm) within the skin. CONCLUSIONS: i) Nanoparticulate encapsulation of OMC increased its "availability" with the SC. ii) The altered distribution of NR when delivered via nanoparticles was due, at least in part, to its altered thermodynamic activity (relative to that in propylene glycol) and, as a result, an increase in its partition coefficient into the SC.

Non-invasive assessment of the effect of formulation excipients on stratum corneum barrier function in vivo.

The objective of the present work was to investigate the effect of formulation excipients on human stratum corneum (SC) barrier function in vivo. Two formulations, an ointment and an oil-in-water cream, were applied to the skin of human volunteers under both occlusive and non-occlusive conditions. The effects of each treatment were then evaluated using three non-invasive biophysical techniques: transepidermal water loss (TEWL), impedance spectroscopy (IS) and attenuated-total-reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. These measurements were combined with a simple tape-stripping protocol to allow information to be derived across the entire SC. IS and TEWL provided basic information on the effect of each formulation on skin barrier function, while ATR-FTIR enabled (i) the tracking of formulation excipients and evaluation of their concentration profiles within the SC, and (ii) deduction of mechanistic detail with which to explain the TEWL and IS results. It was found that occlusion of the skin either in the presence or absence of the cream caused TEWL to be increased when the treatment was terminated at 6 h. Uptake of ointment into the SC, on the other hand, inhibited the post-application TEWL rate. In parallel, treatment with the ointment caused an increase in relative low-frequency skin impedance, consistent with the entry of additional lipophilic constituents into the SC. The latter was confirmed by ATR-FTIR spectroscopic measurements. Overall, the combined use of the three biophysical measurements allowed formulation effects on, and uptake into, the SC to be deduced and evaluated, and the approach may prove useful for the future selection and optimization of topical drug delivery vehicles.

Iontophoretic drug delivery.

The composition and architecture of the stratum corneum render it a formidable barrier to the topical and transdermal administration of therapeutic agents. The physicochemical constraints severely limit the number of molecules that can be considered as realistic candidates for transdermal delivery. Iontophoresis provides a mechanism to enhance the penetration of hydrophilic and charged molecules across the skin. The principal distinguishing feature is the control afforded by iontophoresis and the ability to individualize therapies. This may become significant as the impact of interindividual variations in protein expression and the effect on drug metabolism and drug efficacy is better understood. In this review we describe the underlying mechanisms that drive iontophoresis and we discuss the impact of key experimental parameters-namely, drug concentration, applied current and pH-on iontophoretic delivery efficiency. We present a comprehensive and critical review of the different therapeutic classes and molecules that have been investigated as potential candidates for iontophoretic delivery. The iontophoretic delivery of peptides and proteins is also discussed. In the final section, we describe the development of the first pre-filled, pre-programmed iontophoretic device, which is scheduled to be commercialized during the course of 2004.

Effects of sucrose oleate and sucrose laureate on in vivo human stratum corneum permeability.

PURPOSE: The purpose of this work was to 1) investigate the effect of sucrose esters (sucrose oleate and sucrose laureate in water or in Transcutol, TC) on the stratum corneum (SC) barrier properties in vivo and 2) examine the impact of these surfactant-like molecules on the in vivo percutaneous penetration of a model penetrant 4-hydroxybenzonitrile (4-HB). METHODS: Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and transepidermal water loss measurements were used to evaluate the sucrose oleate- and sucrose laureate-induced biophysical changes in SC barrier function in vivo. In addition. the effect of the enhancers on 4-HB penetration was monitored in vivo using ATR-FTIR spectroscopy in conjunction with tape-stripping of the treated site. RESULTS: Treatment of the skin with 2% sucrose laureate or sucrose oleate in TC significantly increased the extent of 4-HB penetration relative to the control. Furthermore, when skin treated with these formulations was examined spectroscopically, the C-H asymmetric and symmetric stretching bands of the lipid methylene groups were characterized by 1) decreased absorbances and 2) frequency shifts to higher wavenumbers. These effects on the SC lipids and 4-HB penetration were more pronounced for sucrose laureate when combined with TC. CONCLUSIONS: A combination of sucrose esters (oleate or laureate) and TC is able to temporally alter the stratum corneum barrier properties, thereby promoting 4-HB penetration. These molecules are worthy of further investigation as potential candidates for inclusion in transdermal formulations as penetration enhancers.

Occlusive properties of monolayer patches: in vitro and in vivo evaluation.

PURPOSE: Patches can cause a different grade of skin occlusion, depending on matrix composition and thickness, backing layer material. The aim of this work was to verify if in vitro water vapour permeability (WVP) values are predictive of transepidermal water loss (TEWL) and Fourier-transform infrared (FTIR) spectroscopy values measured in vivo after 24 h of methacrylic or acrylic monolayer patches application. The correlation between both in vivo methods has been evaluated. METHODS: The WVP, TEWL and FTIR measurements were performed by using four patches made of a methacrylic or an acrylic polymeric system (250 and 500 microm thickness on a polyurethane backing layer). A fifth patch was made of the methacrylic matrix on a polvvinyl chloride backing layer. RESULTS. A good correlation was found between TEWL values and IR water/lipid absorbance ratios. The in vitro WVP values are in a good correlation with the results of both in vivo methods: TEWL = -().()WVP + 21.31 (R2 = 0.9312): FTIR water/lipid ratio = -0.01WVP + 27.15 (R2 = 0.9447). CONCLUSIONS: The in vitro method proposed for measuring the WVP is predictive of the degree of occlusion resulting from the in vivo application of monolayer patches.