International Journal of Pharmaceutics (v.219, #1-2)

Targeted drug delivery systems 6: Intracellular bioreductive activation, uptake and transport of an anticancer drug delivery system across intestinal Caco-2 cell monolayers by Laxmikant Gharat; Rajneesh Taneja; Natthida Weerapreeyakul; Bhagwant Rege; James Polli; Prashant J Chikhale (1-10).
We demonstrate transport across, intracellular accumulation and bioreductive activation of a conformationally constrained, anticancer drug delivery system (the CH3-TDDS) using Caco-2 cell monolayers (CCMs) as an in vitro model of the human intestinal mucosa. Reverse-phase High Performance Liquid Chromatography (HPLC) coupled with UV detection was used to detect CH3-TDDS, the bioreduction product (lactone) and the released drug (melphalan methyl ester; MME). Upon incubation of the CH3-TDDS with the apical (AP) surface of 21-day-old CCM, we observed rapid decrease in the AP concentration of the CH3-TDDS (60%/hr) as a result of cellular uptake. Rapid intracellular accumulation of the CH3-TDDS was followed by bioreductive activation to deplete the cellular levels of CH3-TDDS. The drug part (MME) and lactone, as well as CH3-TDDS, were detected in the basolateral (BL) chamber. Intracellular Caco-2 levels of TDDS and lactone were also detectable. Bioreductive activation of the CH3-TDDS was additionally confirmed by formation of lactone after incubation of the CH3-TDDS in the presence of freshly prepared Caco-2 cell homogenates. During transport studies of melphalan or MME alone (as control), the intact drug was not detected in the intracellular compartment or in the BL chamber. These observations demonstrate that CH3-TDDS has potential for improving intestinal delivery of MME. TDDS could be useful in facilitating oral absorption of MME as well as the oral delivery of other agents.
Keywords: Anticancer drug delivery system; Caco-2; Intestinal transport; Intracellular bioreductive activation; Melphalan; Uptake;

Effect of ethanol and isopropyl myristate on the availability of topical terbinafine in human stratum corneum, in vivo by Ingo Alberti; Yogeshvar N Kalia; Aarti Naik; Jean-Daniel Bonny; Richard H Guy (11-19).
Purpose: The objective of this study was to determine the availability of the topical drug terbinafine (TBF) in human stratum corneum (SC) in vivo following its administration in formulations containing isopropyl myristate and ethanol. Methods: The ventral forearms of human volunteers were treated for 4 h with TBF, at a concentration equal to 1/4 saturation, in isopropyl myristate (IPM), in ethanol (EtOH) and in 50:50 v/v IPM/EtOH. At the end of the application period, the treated sites were carefully cleaned of excess vehicle and the SC was progressively removed by sequential tape stripping. TBF was quantified in the SC by: (a) extraction of the tape strips and subsequent HPLC analysis; and (b) attenuated total reflectance infrared spectroscopy (ATR-FTIR) of each sequentially exposed SC surface during the tape stripping procedure. Results: The concentration profile of TBF in the SC (i.e. drug concentration as a function of depth in the membrane) was fitted to the appropriate solution of Fick's second law of diffusion, allowing thereby the drug's SC/vehicle partition coefficient (K) and characteristic diffusion parameter (D/L 2, where D is the diffusivity of TBF in the SC of thickness L) to be deduced. Conclusions: While D/L 2 for TBF derived from the three vehicles remained essentially constant, the drug's partitioning into the SC was significantly higher from formulations containing ethanol. Both the semi-quantitative infrared data and the more rigorous HPLC results supported these deductions.
Keywords: Antimycotics; Topical vehicles; Topical administration; Topical bioavailability; Fourier transform infrared spectroscopy; Tape stripping;

Physicochemical assessments of parenteral lipid emulsions: light obscuration versus laser diffraction by David F Driscoll; Frank Etzler; Thomas A Barber; Jorg Nehne; Wilhelm Niemann; Bruce R Bistrian (21-37).
The United States Pharmacopeia (USP) has proposed a new Chapter 〈729〉 entitled ‘Globule Size Distribution in Intravenous Emulsions’ that is intended to identify methods for analyzing the stability of lipid emulsions. We studied the differences between particle-sizing instruments when analyzing the physicochemical stability of a parenteral nutrition mixture compounded with intravenous lipid emulsion, known as an all-in-one mixture. As the growth of lipid droplets, i.e. coalescence, signals an irreversible change in emulsion stability, we focused our investigation on the large diameter tail (>5 μm) of the globule size distribution. Of the four proposed methods, droplet size was studied over a range of mixture stabilities using a low osmolality parenteral nutrition formula employing both light scattering and light obscuration techniques. In addition, the same mixtures were also freshly prepared, and then spiked with a known amount of 5 μm latex spheres. The response obtained from the light obscuration technique was linear and detected both unstable and latex-spiked mixtures in every case for droplets or particles >5 μm. The results of the laser diffraction method were non-linear and overestimated, was less sensitive or missed entirely, globules or particles in the large diameter tail of the dispersion. The results demonstrate that light obscuration is superior to laser diffraction in identifying unstable intravenous fat emulsions.

The effect of hydroxypropylmethylcellulose (HPMC) on the thermal behaviour of nicotinamide was studied. Binary mixtures of nicotinamide and HPMC, composed of various weight fractions of HPMC (X HPMC), were heated, cooled and subsequently re-heated. HPMC dissolved in fused nicotinamide at 140°C. The binary mixture at compositions 0≦X HPMC≦0.3 formed a film structure on cooling. At X HPMC≧0.4, the molten nicotinamide at 140°C was saturated with HPMC. These heated mixtures did not form a homogeneous film by cooling to ambient temperature. At X HPMC<0.4, differential scanning calorimetry peaks originating from recrystallization and melting of nicotinamide were observed in the cooling and re-heating scans, respectively. These peaks became smaller with increase in X HPMC and disappeared at X HPMC∼0.4. Decrease in crystallinity with increase in X HPMC was confirmed by X-ray diffraction. The glass transition temperature of the cooled mixture (T g) increased with increase in X HPMC. When the enthalpy of melting of nicotinamide and 1/T g were plotted against X HPMC, inflections were observed at similar X HPMC values, 0.37–0.38. Dissolution of HPMC in molten nicotinamide was accompanied by hydrogen bond formation, which was confirmed by infrared studies.
Keywords: Nicotinamide; Hydroxypropylmethylcellulose; Differential scanning calorimetry; Hot stage microscopy; Glass transition;

Formulation of a reservoir-type testosterone transdermal delivery system by Mi-Kyeong Kim; Hong Zhao; Chi-Ho Lee; Dae-Duk Kim (51-59).
A reservoir-type transdermal delivery system of testosterone (TS) was developed using an ethanol/water (70:30) cosolvent system as the vehicle. The maximum permeation rate achieved by 70% (v/v) of ethanol was further increased from 2.69 to 47.83 μg/cm2/h with the addition of 1.0% dodecylamine as the skin permeation enhancer. The permeation rate of TS through the ethylene vinyl acetate (EVA) membrane was observed to increase as the vinyl acetate content in the copolymer increased. Addition of 1.0% (w/w) gelling agent, hydroxypropyl methlycellulose (HPMC), in the reservoir formulation resulted in desirable rheological properties with an insignificant effect on the skin permeation rate of TS. Thus, a new transdermal delivery system for TS was formulated using EVA membrane coated with a pressure-sensitive adhesive (Duro-Tak 87-2510) and HPMC as a gelling agent. This experimental patch showed comparable plasma concentration profiles in the in vivo study when compared with a commercial product, Androderm®. Moreover, the results suggested the possibility of further enhancing the permeation rate of TS by controlling the composition of the reservoir formulation.
Keywords: Testosterone; Transdermal; Reservoir-type; Formulation;

Effect of liposomes and niosomes on skin permeation of enoxacin by Jia-You Fang; Chi-Tzong Hong; Wen-Ta Chiu; Ying-Yue Wang (61-72).
The skin permeation and partitioning of a fluorinated quinolone antibacterial agent, enoxacin, in liposomes and niosomes, after topical application, were elucidated in the present study. In vitro percutaneous absorption experiments were performed on nude mouse skin with Franz diffusion cells. The influence of vesicles on the physicochemical property and stability of the formulations were measured. The enhanced delivery across the skin of liposome and niosome encapsulated enoxacin had been observed after selecting the appropriate formulations. The optimized formulations could also reserve a large amount of enoxacin in the skin. A significant relationship between skin permeation and the cumulative amount of enoxacin in the skin was observed. Both permeation enhancer effect and direct vesicle fusion with stratum corneum may contribute to the permeation of enoxacin across skin. Formulation with niosomes demonstrated a higher stability after 48 h incubation compared to liposomes. The inclusion of cholesterol improved the stability of enoxacin liposomes according to the results from encapsulation and turbidity. However, adding negative charges reduced the stability of niosomes. The ability of liposomes and niosomes to modulate drug delivery without significant toxicity makes the two vesicles useful to formulate topical enoxacin.
Keywords: Enoxacin; Liposomes; Niosomes; Skin permeatiom;

Evaluation of creams and ointments as suitable formulations for peldesine by Tacey X Viegas; Lise L Van Winkle; Paul A Lehman; Sue F Franz; Thomas J Franz (73-80).
In-vitro studies were conducted to study the efficacy of mixed and self-emulsifying creams and hydrophobic ointment formulations in delivering peldesine (BCX-34) into and across cryopreserved human cadaver skin (HCS). Oil-in-water cream formulations, containing 1% w/w of radiolabeled C14 BCX-34 and propylene glycol (PG), glycerin (GLY), isopropyl myristate (IPM), oleic acid (OA) and capric-caprylic esters (CE) were prepared. Petrolatum and lanolin based ointments were also prepared with PG. Sections of the HCS, 250 μm thick, were fitted to vertical Franz diffusion chambers containing a receptor medium of pH 7.4 phosphate buffer solution maintained at 37°C. Using the finite dose technique, 4–6 mg of a formulation sample was applied to the epidermal surface of each section and drug diffusion was permitted for 12 and 24 h periods. The distribution of drug into the HCS epidermis, dermis and into the receptor medium was measured by scintillation spectroscopy. The results show good correlation of the calculated in-vitro values for flux and skin-vehicle partition coefficients against the observed amounts of drug detected in the HCS. The mixed emulsion cream formulation containing PG delivered higher amounts of drug into the skin when compared to the same formulation containing GLY cream. The self-emulsifying cream formulation containing IPM had a higher skin-vehicle partition coefficient and delivered more drug into the dermis when compared to those formulations that contained OA and CE. The petrolatum ointment delivered six times more drug into the epidermis than the lanolin ointment, and had higher skin-vehicle partition values. In conclusion, creams containing PG and petrolatum-base formulations would be suitable for BCX-34 dermal delivery.
Keywords: Peldesine; BCX-34; Dermal formulations; In-vitro skin delivery; Purine nucleoside inhibitor; Penetration enhancers;

An oral controlled release matrix pellet formulation containing nanocrystalline ketoprofen by G.J Vergote; C Vervaet; I Van Driessche; S Hoste; S De Smedt; J Demeester; R.A Jain; S Ruddy; J.P Remon (81-87).
A controlled release pellet formulation using a NanoCrystal® colloidal dispersion of ketoprofen was developed. In order to be able to process the aqueous NanoCrystal® colloidal dispersion into a hydrophobic solid dosage form a spray drying procedure was used. The in vitro dissolution profiles of wax based pellets loaded with nanocrystalline ketoprofen are compared with the profiles of wax based pellets loaded with microcrystalline ketoprofen and of a commercial sustained release ketoprofen formulation. Pellets were produced using a melt pelletisation technique. All pellet formulations were composed of a mixture of microcrystalline wax and starch derivatives. The starch derivatives used were waxy maltodextrin and drum dried corn starch. Varying the concentration of drum dried corn starch increased the release rate of ketoprofen but the ketoprofen recovery remained problematic. To increase the dissolution yield surfactants were utilised. The surfactants were either added during the production process of the NanoCrystal® colloidal dispersion (sodium laurylsulphate) or during the pellet manufacturing process (Cremophor® RH 40). Both methods resulted in a sustained but complete release of nanocrystalline ketoprofen from the matrix pellet formulations.
Keywords: Ketoprofen; Sustained release; Nanocrystals; Matrix pellets; Surfactants; Bioavailability;

The influence of vacuum mixing on methylmethacrylate liberation from acrylic cement powder by A Bettencourt; A Calado; J Amaral; F.M Vale; J.M.T Rico; J Monteiro; M Castro (89-93).
Polymethylmethacrylate (PMMA) bone cement is a biomaterial used to anchor prostheses during joint replacement surgery. Residual methylmethacrylate monomer (MMA) may be related with the cytotoxic effect of PMMA. The aim of the present paper was to investigate the effect of two different cement mixing methods: hand stirring at atmospheric pressure and under partial vacuum (0.330 and 0.154 bar) on residual monomer liberation in phosphate buffer saline solution from acrylic cement powder. Residual MMA content was determined by high-performance liquid chromatography. Mathematical models were applied to experimental dissolution data revealing that monomer release was significantly reduced in bone cement powder obtained at 0.154 bar vacuum pressure compared to the other mixing conditions. The kinetic models applied are consistent with a simple diffusion mechanism of the monomer from the polymer matrix.
Keywords: Poly(methylmethacrylate); Methylmethacrylate; Biomaterial; Bone cement; Release model; Vacuum mixing;

Transdermal delivery of naloxone: effect of water, propylene glycol, ethanol and their binary combinations on permeation through rat skin by Ramesh Panchagnula; Pramod Sridhar Salve; Narisetty Sunil Thomas; Amit Kumar Jain; Poduri Ramarao (95-105).
The effect of the solvent systems water, ethanol (EtOH), propylene glycol (PG) and their binary combinations was studied on the ex vivo permeation profile of the opioid receptor antagonist, naloxone, through rat skin. Fourier transform-infrared (FT-IR) spectroscopic studies were done to investigate the effect of enhancers on the biophysical properties of the stratum corneum (SC), in order to understand the mechanism of permeation enhancement of naloxone by the solvent systems used. The flux of naloxone was found to increase with increasing concentrations of EtOH, upto 66% in water, and PG upto 50% in water. The maximum flux of 32.85 μg cm−2 h−1 was found with 33% PG in EtOH. The FT-IR spectra of SC treated with EtOH showed peak broadening at 2920 cm−1 at all concentrations of EtOH studied indicating that EtOH increases the translational freedom (mobility) of lipid acyl chains. Theoretical blood levels well above the therapeutic concentration of naloxone can be achieved with the solvent system comprising 33% PG in EtOH and hence, provides flexibility in choice of patch size depending on the addiction status of the patient to be treated.
Keywords: Naloxone; Transdermal permeation; Solvent system; Fourier transform-infrared spectroscopy;

Electric field analysis on the improved skin concentration of benzoate by electroporation by Kenji Sugibayashi; Maki Yoshida; Kenji Mori; Tetsuya Watanabe; Tetsuya Hasegawa (107-112).
The objective in the present study was to understand the relationship between the increased skin concentration of benzoate as a model drug after topical application of its sodium salt and the electric field intensity produced in the skin barrier, the stratum corneum, by electroporation. A piece of excised abdominal hairless rat skin was set in a Franz type diffusion cell, and 0.5% sodium benzoate and physiological saline were applied to the stratum corneum and dermis sides, respectively. Two needle electrodes made of Ag were connected to an electrical power source, which produced exponentially decaying pulses. The electrodes were placed on the skin surface with a distance of 0.5 cm between both electrodes. After the 4 h passive permeation experiment, an electrical pulse was applied to the rat skin at 300 V every minute for 10 min. The skin was then removed from the diffusion cell, and the amounts of benzoate in different positions of the skin specimen were measured. Field intensity generated in the stratum corneum by electroporation was determined by a finite element method using a computer program. The amounts of benzoate at different sites in the skin were almost proportional to the mean field intensity in the corresponding stratum corneum. These results suggested that the enhancing effect of electroporation can be evaluated by the field intensity more directly than the application voltage.
Keywords: Skin permeation; Skin concentration; Electroporation; Electric field analysis;

Drying behaviour of two sets of microcrystalline cellulose pellets by Jonas Berggren; Göran Alderborn (113-126).
The objective was to study contraction and densification of two sets of microcrystalline cellulose pellets, prepared using water (W) or a 25/75% w/w water/ethanol (W/E) mixture, during drying. The pellets were dried on microscope slides, photographed and weighed at set times. The porosity of the dry pellets was determined by mercury pycnometry. From pellet size, weight and porosity data, contraction and densification of the pellets and the relationship of these to the liquid content of the pellets during drying were calculated. Both types of pellets contracted and densified during drying. The initial porosity was similar for both types, but the final porosity of the dry pellets was higher for the W/E pellets. Thus, the difference in final pellet porosity between the two types was caused by a difference in densification during drying rather than a different degree of densification during the pelletisation procedure. The contraction rate and the relationships between contraction and the volume of removed liquid, and contraction and the degree of liquid saturation differed between the two types of pellet. The difference in drying behaviour between the two types of pellets can be explained by a liquid related change in both contraction driving force and contraction counteracting force or by a different contraction mechanism.
Keywords: Microcrystalline cellulose pellets; Granulation; Drying; Contraction; Densification; Porosity;

Gelatin microparticles were prepared by co-lyophilization with poly(ethylene glycol) (PEG) as a protein micronization adjuvant. Aqueous solutions containing gelatin and PEG at various mixing ratios were freeze-dried. The lyophilizates were dispersed in methylene chloride and subjected to particle size analysis. The particle size decreased as the PEG/gelatin ratio increased. The microparticles isolated from the suspension had spherical microdomains with sizes ranging from 1 to 10 μm, which indicated that phase separation between PEG and gelatin during freezing was involved in the formation mechanism of gelatin microparticles. By using this technology, gelatin microparticles with an average size of less than 10 μm, with high purity of more than 90% and with good dispersibility could be obtained with high yield. The gelatin microparticles with average sizes from 5 to 20 μm were applied to encapsulation into biodegradable PLGA/PLA microspheres via a solid-in-oil-in-water emulsion process. The entrapment efficiency was highly dependent on the particle size and the size distribution, signifying that solid microparticles with an average diameter of less than 5 μm and an maximal diameter of less than 10 μm would be required for effective encapsulation. These gelatin microparticles would be useful for studying and developing various drug delivery systems.
Keywords: Gelatin microparticles; Freeze-drying; Poly(ethylene glycol); Phase separation; Biodegradable microspheres; Solid-in-oil-in-water emulsion;

Liposomal gels for vaginal drug delivery by Željka Pavelić; Nataša Škalko-Basnet; Rolf Schubert (139-149).
The aim of our study was to develop a liposomal drug carrier system, able to provide sustained and controlled release of appropriate drug for local vaginal therapy. To optimise the preparation of liposomes with regards to size and entrapment efficiency, liposomes containing calcein were prepared by five different methods. Two optimal liposomal preparations (proliposomes and polyol dilution liposomes) were tested for their in vitro stability in media that simulate human vaginal conditions (buffer, pH 4.5). To be closer to in vivo application of liposomes and to achieve further improvement of their stability, liposomes were incorporated in vehicles suitable for vaginal self-administration. Gels of polyacrylate were chosen as vehicles for liposomal preparations. Due to their hydrophilic nature and bioadhesive properties, it was possible to achieve an adequate pH value corresponding to physiological conditions as well as desirable viscosity. In vitro release studies of liposomes incorporated in these gels (Carbopol 974P NF or Carbopol 980 NF) confirmed their applicability as a novel drug carrier system in vaginal delivery. Regardless of the gel used, even 24 h after the incubation of liposomal gel in the buffer pH 4.5 more than 80% of the originally entrapped substance was still retained.
Keywords: Liposomes; Polyacrylate gels; Stability; Vaginal application; Viscosity;

A cylindrical dosage form comprising a laminated composite polymer core and a hydrophobic polycarbonate coating was proposed for programmable drug delivery. In the core, poly[(ethyl glycinate) (benzyl amino acethydroxamate) phosphazene] was synthesized as drug-loaded layers for its strong pH-sensitive degradation (eroded after 1.5 days at pH 7.4 and more than 20 days at pH 5.0 and 6.0). Poly(sebacic anhydride)-b-polyethylene glycol or poly(sebacic anhydride-co-trimellitylimidoglycine)-b-poly(ethylene glycol) was selected as isolating layers for their good processing properties at room temperature and suitable erosion duration. The in vitro drug release studies of these devices were conducted under physiological conditions (pH 7.4). The results revealed that the model drugs (brilliant blue, FITC-dextran, myoglobin) could be released in typical pulsatile manner. Moreover, the duration time of drug release (24–40 h) and the lag time (18–118 h) could be separately regulated by the mass of polyphosphazene and the type or mass of polyanhydride. In this experiment, the cooperative effect of polyanhydrides and pH-sensitive degradable polyphosphazene was specially demonstrated, which offers a new idea to develop a programmable drug delivery system for single dose vaccine and other related applications.
Keywords: Programmable drug delivery; Polyanhydride; Polyphosphazene;

Transdermal permeation of propranolol through human skin in the presence of fatty acid (lauric, capric) penetration enhancers has been investigated. Thermal analysis showed that binary mixtures of propranolol with either fatty acid were not simple mechanical mixtures of the two components. Propranolol formed 1:1 molar addition compounds with both lauric and capric acids; the addition compound produced from propranolol and lauric acid (m.p. 79°C) also developed eutectic systems with both propranolol (m.p. 54°C) and lauric acid (m.p. 16°C). Similarly, the addition compound made from propranolol and capric acid (m.p. 97°C) formed eutectic systems with propranolol (m.p. 83°C) and capric acid (m.p. 15°C). Infrared analyses indicated that the addition compounds were fatty acid salts of the β-blocker. The nature of the species permeating through human epidermal membranes from binary mixtures of propranolol with the fatty acids was investigated using a novel attenuated total reflectance Fourier transform infrared method. There was no clear difference in permeation rates of the fatty acids compared with the β-blocker, suggesting that the permeating species was the intact addition compound. The influence of melting point depression of the β-blocker fatty acid systems on transdermal permeation was predicted from a mathematical model; predicted and experimentally determined data correlated well thus providing an alternative explanation as to the mode of action of these permeation enhancers.
Keywords: Propranolol; Fatty acids; Attenuated total reflectance Fourier transform infrared; Transdermal permeation; Thermal analysis; Eutectic;

The effect of penetration enhancer (i.e., 1, 2, 3 and 5% menthone in combination with 50% ethanol (EtOH)) was investigated on the in vitro percutaneous absorption of tamoxifen, and post-recovery epidermal permeability after removal of the above enhancer. The flux of tamoxifen with menthone in combination with 50% EtOH was significantly greater (P<0.05) than the control (50% EtOH). The flux of tamoxifen increased with increasing concentrations of menthone. The post-recovery flux through enhancer exposed epidermis was significantly decreased (P<0.05) as compared to pre-recovery. However, post-recovery flux of tamoxifen through the enhancer-exposed epidermis did not completely recover to the baseline (i.e., post-recovery flux through phosphate buffered saline, pH 7.4 treated epidermis).
Keywords: Penetration enhancer; Menthone; Percutaneous absorption; Tamoxifen; Skin reversibility;

Notice board (185-188).