International Journal of Pharmaceutics (v.200, #2)

Studies on the cyclosporin A loaded stearic acid nanoparticles by Qiang Zhang; Guoqing Yie; Yie Li; Qingsong Yang; T Nagai (153-159).
Stearic acid nanoparticles were prepared in this study by melt-homogenization to investigate the possibility of them as a new kind of drug carrier system. Some physicochemical properties of stearic acid nanoparticles were studied and morphology examined by transmission electron microscope. Cyclosporin A as a model drug was then encapsulated into stearic acid nanoparticles. Following the establishment of high performance liquid chromatography assay for cyclosporin A analysis in stearic acid nanoparticles or blood samples, the encapsulation ratio of cyclosporin A to stearic acid nanoparticles was estimated and pharmacokinetics as well as bioavailability of cyclosporin A stearic acid nanoparticles after oral administration to Wistar rats were studied, using the Sandimmun Neoral® (an available microemulsion system of cyclosporin A) as a reference. The mean diameter of cyclosporin A stearic acid nanoparticles was 316.1 nm, while the encapsulation ratio of cyclosporin A to stearic acid nanoparticles reached to 88.36%. It was demonstrated by IR spectra and differential scanning calorimetry that there was no chemical reaction occurred between the cyclosporin A and stearic acid. The relative bioavailability of cyclosporin A stearic acid nanoparticles over reference was nearly 80%, and the time to reach maximum concentration (T max) of cyclosporin A after oral administration of cyclosporin A stearic acid nanoparticles was delayed significantly than the reference, suggesting an obvious sustained release effect. The stearic acid nanoparticles might be a very potential drug carrier.
Keywords: Drug carrier; Stearic acid; Nanoparticles; Cyclosporin A; Relative bioavailability;

The present study was conducted to obtain a deeper insight into the mechanism of drug release from HPMC matrices. The microstructure, mobility, internal pH and the state of water within the gel layer of hydrated HPMC matrices (having different molecular weights) containing naproxen sodium (NS) and naproxen (N) were studied using Electron Paramagnetic Resonance (EPR), Nuclear Magnetic Resonance (NMR) and Differential Scanning Calorimetry (DSC) techniques. The study show that matrices composed of various viscosity grades of HPMC are characterized by similar microviscosity values in spite of the difference in their molecular weight. The NMR and DSC results led to the conclusion that higher molecular weights of HPMC are characterized by higher water absorption capacity and higher swelling. Analysis of non-freezable water in HPMC(K4M)–NS system revealed that addition of NS to solution increased the fraction of water bound to K4M+NS compared with the equivalent solutions without NS. The results suggest that the drug is participating in the crystallization of water and leads to the formation of a three dimensional network structure that decreases the freedom of water in K4M+NS samples. Calculation of the number of hydration shells showed that up to 2.2 layers are involved in HPMC-NS hydration compared to 1.5 layers for HPMC gel without NS. This was explained based on the different water ordering in the gel induced by NS as results of its absorption to polymer surface. Microviscosity values measured by EPR for K4M/N and K4M/NS hydrated matrices were found to be higher for K4M/N matrices, especially at initial stage of hydration. Mobile compartment calculations showed lower values for K4M/N compared with K4M/NS matrices. pH measurements by EPR revealed that incorporation of N to HPMC matrix led to lower internal pH value inside the hydrated tablet compared with NS. This behavior led to lower solubility of N which dictates its surface erosion mechanism, compared with NS matrix that was characterized by higher internal pH value and higher drug solubility. These properties of HPMC/NS increased chain hydration and stability, and led to drug release by the diffusion mechanism.
Keywords: Hydroxypropyl methylcellulose; Naproxen; Hydration; Electron paramagnetic resonance; Nuclear magnetic resonance; Differential scanning calorimetry;

In the search of new prodrugs effective against herpes simplex virus series of thymidine, 5-bromo-2′-deoxyuridine esters with amino acid and peptide chains and 3′-azido-2′,3′-dideoxythymidine derivatives have been synthesized and evaluated for antiviral activity. The chemical stability of some of them containing different residues was studied at pH 1 and 7.4 and temperature of 37°C. An HPLC method was developed for quantification of the unchanged ester concentration. It was proved that esters with simple aliphatic straight side chain (containing alanyl-, glycyl-, or glycyl-glycyl-glycyl-residues) are relatively stable both at acidic and neutral media, 37°C. Some of them undergo negligible hydrolysis with half lifes ranging between 6 and 23 h. In contrast, more complex esters with branched side chain (valyl-), with phenyl residue (phenylalanyl-), as well as containing thiazol ring are rather unstable especially at acidic conditions and undergo rapid hydrolysis resulting in the respective chemical precursor. The stability of the former group esters outlines them as suitable candidates for prodrugs: with higher lipophilicity facilitating po absorption, satisfying chemical stability and possibility to release the active moiety following enzymatic hydrolysis.
Keywords: Prodrugs; Pyrimidine 2′-deoxynucleosides; Amino acids; Peptides;

Stability of thioTEPA and its metabolites, TEPA, monochloroTEPA and thioTEPA-mercapturate, in plasma and urine by Maria J van Maanen; Karen Doesburg Smits; J.Mirjam A Damen; Albert J.R Heck; Jos H Beijnen (187-194).
The degradation of N,N′,N′′-triethylenethiophosphoramide (thioTEPA) and its metabolites N,N′,N′′-triethylenephosphoramide (TEPA), N,N′-diethylene,N′′-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA-mercapturate in plasma and urine has been investigated. ThioTEPA, TEPA and monochloroTEPA were analyzed using a gas chromatographic (GC) system with selective nitrogen/phosphorous detection; thioTEPA-mercapturate was analyzed on a liquid chromatography-mass spectrometric (LC-MS) system. The influences of pH and temperature on the stability of thioTEPA and its metabolites were studied. An increase in degradation rate was observed with decreasing pH as measured for all studied metabolites. In urine the rate of degradation at 37°C was approximately 2.5±1 times higher than at 22°C. At 37°C thioTEPA and TEPA were more stable in plasma than in urine, with half lives ranging from 9–20 h for urine and 13–34 h for plasma at pH 6. Mono- and dichloro derivatives of thioTEPA were formed in urine and the monochloro derivative was found in plasma. Degradation of TEPA in plasma and urine resulted in the formation of monochloroTEPA. During the degradation of TEPA in plasma also the methoxy derivative of TEPA was formed as a consequence of the applied procedure. The monochloro derivative of thioTEPA-mercapturate was formed in urine, whereas for monochloroTEPA no degradation products could be detected.
Keywords: ThioTEPA; TEPA; MonochloroTEPA; ThioTEPA-mercapturate; Stability in plasma and urine;

An in vivo investigation of the rabbit skin responses to transdermal iontophoresis by Angela Anigbogu; Sunita Patil; Parminder Singh; Puchun Liu; Steven Dinh; Howard Maibach (195-206).
To optimize the benefits of transdermal iontophoresis, it is necessary to develop a suitable animal model that would allow for extensive assessments of the biological effects associated with electro-transport. Rabbit skin responses to iontophoresis treatments were evaluated by visual scoring and by non-invasive bioengineering parameters and compared with available human data. In the current density range 0.1–1.0 mA/cm2 applied for 1 h using 0.9% w/v NaCl and 0.5 mA/cm2 for up to 4 h, no significant irritation was observed. 2 mA/cm2 applied through an area of 1 cm2 for 1 h resulted in slight erythema at both active electrode sites but without significant changes in transepidermal water loss (TEWL) and laser Doppler velocimetry (LDV). A value of 4 mA/cm2 under similar conditions caused moderate erythema at the anode and cathode with TEWL and LDV being significantly elevated at both sites; 1 mA/cm2 current applied for 4 h, caused moderate erythema at both anode and cathode; and 1 mA/cm2 applied for 1 h caused no irritation when the area of exposure was increased from 1 to 4.5 cm2. When significant irritation and barrier impairment occurred, the erythema was resolved within 24 h with barrier recovery complete 3–5 days post-treatment. Rabbit skin thus shows promise as an acceptable model for iontophoresis experiments.
Keywords: Iontophoresis; Skin; Irritation; erythema; Transepidermal water loss; Laser Doppler velocimetry;

Characterization of a diltiazem-lambda carrageenan complex by M.C Bonferoni; S Rossi; F Ferrari; G.P Bettinetti; C Caramella (207-216).
In the present paper the interaction between lambda carrageenan, a natural sulphated polysaccharide, and diltiazem HCl, a Ca channel blocking agent, was studied. Dialysis equilibria were performed to quantify the binding capacity of lambda carrageenan for diltiazem. The relevance of the interaction to hydrophilic matrix systems was confirmed: a relationship was found between the binding capacity and the release profiles of matrix tablets containing a fixed amount of drug and different percentages of lambda carrageenan. Dialysis equilibria in buffered media showed that the interaction is quite insensitive to the pH of the medium (in the range 1.8–6.8), while it is reduced by increasing ionic strength; this behaviour is in line with the importance of ionic bonds in diltiazem-carrageenan interaction. On the basis of the calculated binding capacity, the complex was prepared, dried and milled. A preliminary characterization of the diltiazem-carrageenan complex in the solid state was effected by means of X-ray and DSC analysis. The amount of drug going into solution from the complex was not significantly affected by the pH of the medium (in the range 1.8–6.8), while it was increased by increasing ionic strength.
Keywords: Lambda carrageenan; Drug–polymer complex; Dialysis equilibria; Diltiazem HCl; Controlled release matrix tablets;

The molar solubilization capacities (κ) and the molar micelle–water partition coefficients (K M N) in Polysorbate 80 of several drugs (including barbiturates, steroids, and benzoic acid derivatives) are related to their log octanol–water partition coefficients (log  P). Both κ and K M N values were calculated from solubility versus Polysorbate 80 concentration profiles, which were either experimentally determined or obtained from the literature. There is a linear relationship between log  P of the tested compounds and the logarithm of the molar micelle–water partition coefficient (log  K M N). On the other hand molar solubilization capacities are nearly independent of log  P. It is shown that the ability of Polysorbate 80 to solubilize a drug can be predicted from its log  P value.
Keywords: Polysorbate 80; Octanol–water; Solubilization descriptors; Partition coefficients;

In vitro evaluation of sol–gel processed spray dried silica gel microspheres as carrier in controlled drug delivery by Pirjo Kortesuo; Manja Ahola; Minna Kangas; Ilkka Kangasniemi; Antti Yli-Urpo; Juha Kiesvaara (223-229).
The objective of this study was to evaluate sol–gel-derived spray dried silica gel microspheres as carrier material for dexmedetomidine HCl and toremifene citrate. The drug was dissolved in sol–gel processed silica sol before spray drying with Büchi laboratory scale equipment. Microspheres with a low specific surface area were spherical by shape with a smooth surface without pores on the external surface. The particle size distribution was quite narrow. The in vitro release of toremifene citrate and dexmedetomidine HCl showed a dose-dependent burst followed by a slower release phase, that was proportional to the drug concentration in the concentration range between 3.9 and 15.4 wt.%. The release period for toremifene citrate was approximately 10 days and for dexmedetomidine HCl between 7 and 50 days depending on drug concentration. Spray drying is a promising way to produce spherical silica gel particles with a narrow particle size range for controlled delivery of toremifene citrate and dexmedetomidine HCl.
Keywords: Spray drying; Drug release; Silica gel microspheres; Dexmedetomidine HCl; Toremifene citrate;

Clonazepam release from core-shell type nanoparticles of poly(ε-caprolactone)/poly(ethylene glycol)/poly(ε-caprolactone) triblock copolymers by Jae-Gon Ryu; Young-Il Jeong; In-Sook Kim; Jong-Hoon Lee; Jae-Woon Nah; Sung-Ho Kim (231-242).
The triblock copolymer based on poly(ε-caprolactone) (PCL) as hydrophobic part and poly(ethylene glycol) (PEG) as hydrophilic one was synthesized and characterized. Core-shell type nanoparticles of poly(ε-caprolactone)/poly(ethylene glycol)/poly(ε-caprolactone) (CEC) block copolymer were prepared by a dialysis technique. According to the amphiphilic characters, CEC block copolymer can self-associate at certain concentration and their critical association concentration (CAC) was determined by fluorescence probe technique. CAC value of the CEC-2 block copolymer was evaluated as 0.0030 g/l. CAC values of CEC block copolymer decreased with the increase of PCL chain length, i.e. the shorter the PCL chain length, the higher the CAC values. From the observation of transmission electron microscopy (TEM), the morphologies of CEC-2 core-shell type nanoparticles were spherical shapes. Particle size of CEC-2 nanoparticles was 32.3±17.3 nm as a monomodal and narrow distribution. Particle size, drug loading, and drug release rate of CEC-2 nanoparticles were changed by the initial solvents and the molecular weight of CEC. The degradation behavior of CEC-2 nanoparticles was observed by 1H NMR spectroscopy. It was suggested that clonazepam (CNZ) release kinetics were dominantly governed by diffusion mechanism.
Keywords: Clonazepam release; Poly(ε-caprolactone); Poly(ethylene glycol); Poly(ε-caprolactone);

pH, pK a and dermal delivery by Jonathan Hadgraft; Claudia Valenta (243-247).
The effect of pH on the permeation of ibuprofen and lignocaine through human skin has been modelled using a modification to the equation derived by Potts and Guy, which is normally applied to unionized entities. The results show that permeation is related to the distribution coefficient. The physicochemical properties have been predicted ab initio using commercially available software and compared to literature values. The approach is successful and shows that there is significant permeation of the ionized drugs through a lipophilic pathway, possibly as a result of ion pairing. Since the aqueous solubility of the ionized material is significantly higher than the unionized, the maximum flux through the skin may occur at a pH where ionization is high. Optimum topical or transdermal formulations may not therefore be for the free acid or free base.
Keywords: Dermal delivery; Ibuprofen; Lignocaine; pH partition;

The stability of submicron emulsions of different local anesthetic/analgesic substances was investigated in the presence and absence of different hydrophobic excipients (ripening inhibitors). Ostwald ripening was believed to be the underlying mechanism for the instability of these emulsions. In the absence of ripening inhibitors, the mean droplet size of the emulsions increased from 100 nm to about 4–5 μm within an hour of manufacture. The addition of a small amount of a second component of lower solubility to the disperse phase decreased the rate of Ostwald ripening, producing good stability of the emulsions. The efficiency of the ripening inhibitors was directly proportional to their solubility in the disperse phase, i.e. the water. The lower the solubility, the more effective the stabilization of the emulsions. The experimentally observed rates of increase in droplet size in the emulsions were closely correlated with those predicted according to the Liftshitz–Slezov–Wagner (LSW) theory.
Keywords: Local anesthetics; Submicron emulsion stability; Ostwald ripening; Ripening inhibitors;

The objective of the present study was to characterise the TR146 cell culture model as an in vitro model of human buccal mucosa with respect to the enzyme activity in the tissues. For this purpose, the contents of aminopeptidase, carboxypeptidase and esterase in homogenate supernatants of the TR146 cell culture model, and human and porcine buccal epithelium were compared. The esterase activity in the intact cell culture model and in the porcine buccal mucosa was compared. Further, the TR146 cell culture model was used to study the permeability rate and metabolism of leu-enkephalin. The activity of the three enzymes in the TR146 homogenate supernatants was in the same range as the activity in homogenate supernatants of human buccal epithelium. In the TR146 cell culture model, the activity of aminopeptidase (13.70±2.10 nmol/min per mg protein) was approx. four times the activity of carboxypeptidase (3.73±0.53 nmol/min per mg protein), whereas the level of esterase activity was significantly higher (223.39±69.82 nmol/min per mg protein). In the TR146 cell culture model, the apical esterase activity was found significantly higher than the basal activity, and found comparable to the porcine buccal mucosa. However, the esterase activity on the serosal side of the porcine buccal mucosa was higher than in the TR146 cell culture model. Approx. 1.5% of leu-enkephalin permeated the TR146 cell layers within 5 h (P app 7.38±0.83×10−7 cm/s) and approx. 77% of intact peptide was still present in the donor phase after 5 h. The present study suggests that the TR146 cell culture model is a valuable in vitro model for permeability and metabolism studies with enzymatically labile drugs, such as leu-enkephalin, intended for buccal drug delivery.
Keywords: TR146 cell culture model; Human; Porcine; Buccal enzyme activity; Leu-enkephalin; Aminopeptidase; Carboxypeptidase; Esterase;

An atomic force microscopy investigation of bioadhesive polymer adsorption onto human buccal cells by Dharmendra Patel; James R Smith; Andrew W Smith; Nigel Grist; Paul Barnett; John D Smart (271-277).
Atomic force microscopy (AFM) was used to examine the buccal cell surface in order to image the presence of adsorbed bioadhesive polymers identified from previous work. Isotonic saline solution (5 ml) containing either polycarbophil (pH 7.6), chitosan (pH 4.5) or hydroxypropyl methylcellulose (pH 7.6) (0.5% w/v) was exposed to freshly collected buccal cells (ca. 48×104 cells/test) for 15 min at 30°C. The cells were then rinsed with a small volume of double distilled water, allowed to air-dry on a freshy cleaved mica surface and imaged using contact mode AFM. Untreated cells showed relatively smooth surface characteristics, with many small ‘crater-like’ pits and indentations spread over cell surfaces. Cells that had been treated with all the investigated polymers appeared to have lost the crater and indentation characteristic and gained a higher surface roughness. These results suggest that polymer chains had adsorbed onto the cell surfaces. Quantitative image analysis of cell topography showed significant increases (P<0.05) in arithmetic roughness average (R a) for all the investigated polymer treated cells surfaces with respect to untreated control specimens. The changes in surface topography indicate the presence of adsorbed polymer, confirming previous work. This study demonstrates the suitability of AFM as a powerful and sensitive technique for detecting and imaging bioadhesive polymers present on mucosal cell surfaces.
Keywords: Atomic force microscopy; Bioadhesion; Mucoadhesion; Oral cavity; Polymers;

The physico-chemical properties of salmeterol and fluticasone propionate in different solvent environments by Yonatan Michael; Babur Z Chowdhry; Ian C Ashurst; Martin J Snowden; Craig Davies-Cutting; Steven Gray (279-288).
The physico-chemical properties of two anti-asthmatic drugs, salmeterol xinafoate and fluticasone propionate, have been studied in both aqueous and non-aqueous solvent environments. Ultraviolet-visible (UV-Vis) spectroscopy, fluorescence spectroscopy and electrospray ionisation mass spectrometry (ESI-MS) have been used to characterise the interaction of the drugs in 70:30 (v/v) methanol/water solutions. First derivative UV-Vis spectra measurements indicate that an interaction takes place between the two drugs in a binary solvent system. Fluorescence studies indicate that an increase in the concentration of fluticasone propionate results in a decrease in the fluorescence signal of the salmeterol for mixed solutions of the drugs. Analysis of a mixture of the two drug solutions using mass spectrometry also shows evidence of salmeterol-fluticasone propionate interaction and dimer formation with respect to both the salmeterol and the fluticasone propionate. Model metered dose inhalers (MDI) of both individual samples and mixtures of the drugs were formulated as suspensions in solvent CFC-113. The extent of deposition onto different inhaler components, such as the aluminium alloy canister, Teflon coated canister and the metering valve was evaluated by high-performance liquid chromatography (HPLC) of the methanol/water washings of the deposited drug(s). Changing the nature of the surface properties of the container resulted in a significant difference in the extent of deposition. The deposition of the individual drugs was found to increase as the dispersion concentration of the drug increases. However, the formulation based on a combination of the two drugs was found to show different deposition behaviour compared to the individual drug formulations. The deposition of the drugs, onto the aluminium alloy canister and the metering valve, decreases as the combined dispersion concentration of the two drug increases.
Keywords: Salmeterol xinafoate; Fluticasone propionate; Methanol/water; CFC-113; Deposition; UV-Vis; Fluorescence; ESI-MS; HPLC;

Index (289-291).