International Journal of Pharmaceutics (v.194, #1)

In the 1990s chitosan turned out to be a useful excipient in various pharmaceutical formulations. By modifications of the primary amino group at the 2-position of this poly(β1→4 d-glucosamine), the features of chitosan can even be optimised according to a given task in drug delivery systems. For peroral peptide delivery these tasks focus on overcoming the absorption (I) and enzymatic barrier (II) of the gut. On the one hand, even unmodified chitosan proved to display a permeation enhancing effect for peptide drugs. On the other hand, a protective effect for polymer embedded peptides towards degradation by intestinal peptidases can be achieved by the immobilisation of enzyme inhibitors on the polymer. Whereas serine proteases are inhibited by the covalent attachment of competitive inhibitors such as the Bowman–Birk inhibitor, metallo-peptidases are inhibited by chitosan derivatives displaying complexing properties such as chitosan-EDTA conjugates. In addition, because of the mucoadhesive properties of chitosan and most of its derivatives, a presystemic metabolism of peptides on the way between the dosage form and the absorption membrane can be strongly reduced. Based on these unique features, the co-administration of chitosan and its derivatives leads to a strongly improved bioavailability of many perorally given peptide drugs such as insulin, calcitonin and buserelin. These polymers are therefore useful excipients for the peroral administration of peptide drugs.
Keywords: Chitosan; Chitosan derivatives; Peroral peptide application; Drug delivery systems;

3′-Deoxyribonucleosides and their derivatives as anti-amoebic agents by S Ghosal; B.N KrishnaPrasad; M Khanna; A.K Dwivedi; S Singh; A Kumar; S.B Katti (15-20).
In general nucleoside analogues were found to possess in vitro amoebicidal property against trophozoites of Entamoeba histolytica. The acid-labile nature of these compounds completely destroyed their ability to cure caecal amoebiasis of rats. However the therapeutic efficacy of one of these compounds yielded most promising results, with 10% cures when it was administered as enteric coated formulation.
Keywords: Anti-amoebic agents; Caecal amoebiasis of rats; Entamoeba histolytica; Nucleosides;

Formation of HDL-like complexes from apolipoprotein A-IM and DMPC by Malin Suurkuusk; Satish K Singh (21-38).
Conditions for the preparation of reconstituted high density lipoproteins (HDLs) by incubation of the synthetic lipid dimyristoylphosphatidylcholine (DMPC) and recombinant apolipoprotein A-IM have been investigated as a function of ratio of incubation lipid to protein, incubation temperature and the lipid form (multilamellar (MLV) or small unilamellar (SUV) vesicles). The size distributions of the resultant lipid-protein complex particles from various incubations have been evaluated by native gel electrophoresis. Structural changes of the protein after incorporation into these complex particles have been estimated by CD. Thermal characteristics of the particles has been examined by DSC and correlated with CD results. Titration calorimetry has been used to obtain interaction parameters based on a simplified binding model. It is hypothesized that the major enthalpic step in the production of rHDLs is the primary association step between protein and lipid vesicles. It has been shown that by raising the temperature and incubation ratio, the formation of rHDL particles can be directed towards smaller size and a narrower size distribution. The results have been described on the basis of a model where formation of discoidal particles requires prior saturation of vesicle surface area by adsorbed protein, thus explaining differences between particles formed from MLVs and SUVs.
Keywords: Apolipoprotein A-IM; Reconstituted lipoprotein; Phospholipid–protein complex; Differential scanning calorimetry; Titration calorimetry; Circular dichroism;

In vitro assessment of archaeosome stability for developing oral delivery systems by Girishchandra B Patel; Brian J Agnew; Lise Deschatelets; L.Perry Fleming; G.Dennis Sprott (39-49).
The in vitro stability of archaeosomes made from the total polar lipids of Methanosarcina mazei, Methanobacterium espanolae or Thermoplasma acidophilum, was evaluated under conditions encountered in the human gastrointestinal tract. At acidic pH, multilamellar vesicles (MLV) prepared from T. acidophilum lipids were the most stable, releasing ∼80, 20, 10 and 5% of encapsulated 14C-sucrose at pH 1.5, 2.0, 2.5 and 6.2, respectively, after 90 min at 37°C. Archaeosomes from M. mazei lipids were the least stable. For each type of total polar lipid, unilamellar vesicles (ULV) were less stable than the corresponding MLV vesicles. Pancreatic lipase had relatively minor effect on the stability of archaeosomes made from either of the three types of total polar lipids, causing the release of 12–27% of the encapsulated 5(6)-carboxyfluorescein (CF) from ULV and MLV after 90 min at 37°C. In simulated human bile at pH 6.2, MLV from M. mazei total polar lipids lost 100% of the encapsulated CF after 90 min at 37°C, whereas those from the polar lipids of M. espanolae or T. acidophilum lost ∼85% of the marker. Pancreatic lipase and simulated human bile had no synergistic effect on the release of carboxyfluorescein from ULV or MLV prepared from any of the total polar lipids. After 90 min in the combined presence of these two stressors at pH 6.2, the leakage of fluorescein conjugated bovine serum albumin from MLV prepared from T. acidophilum lipids was similar to that of CF, and 13% of the initially present vesicles appeared to be intact. These results indicate that archaeosomes show stability properties indicative of potential advantages in developing applications as an oral delivery system.
Keywords: Archaeal lipid liposomes; Archaeosomes; Stability; Oral delivery system; GI tract;

A laminated device comprising of polyanhydrides as isolating layers and pH-sensitive complexes as protein-loaded layers was designed to deliver proteins in a pulsatile manner. Poly(sebacic anhydride)-b-polyethylene glycol (PSA-b-PEG) and poly(trimellitylimidoglycine-co-sebacic anhydride)-b-polyethylene glycol (P(TMA-gly-co-SA)-b-PEG) were synthesized as isolating layers for their good processing properties at room temperature and suitable erosion duration. During the erosion period, pH of the dissolution fluid decreases to a low value (3.8–5.8). Poly(methacrylic acid)/polyethoxazoline (PMAA/PEOx) complex was used as protein-loaded layers, which could dissociate and release model proteins, Myoglobin (Mb) and Bovine Serum Albumin (BSA), at pH 7.4 while become stable and retained the drugs below pH 5.0. The protein release from the device showed a typical pulsatile fashion. The lag time prior to the pulsatile protein release correlated with the hydrolytic duration of the polyanhydrides, which varied from 30 to 165 h by selecting polyanhydride type and isolating layer thickness. In addition, the pulse duration could be adjusted from 18.5 to 40 h by varying the mass of the complex. The results can be attributed to the synergistic effects between the degrading polyanhydrides, pH-sensitive complexes and proteins.
Keywords: Polyanhydrides; pH-sensitive complexes; Pulsatile protein release; Synergistic effect;

Synthesis and characterisation of a new class of stable S-adenosyl-l-methionine salts by A. Morana; I. Di Lernia; M. Cartenı̀; R. De Rosa; M. De Rosa (61-68).
S-adenosyl-l-methionine (SAM) is an important metabolic intermediate that serves as a donor of methyl and aminopropyl groups to a variety of acceptor molecules. The molecule in vitro is unstable both in solution and in crystalline form undergoing irreversible conversion to 5′-methyltioadenosine (MTA) and homoserine lactone. Since this form of instability seems to be prevented in the cell of the living organism by bonds with macromolecules, we designed and developed a novel class of salts of SAM with large size anions to improve the stability of the sulfonium compound outside the cell. For this purpose we synthesised and characterised by NMR and IR spectroscopy anions consisting of amidic derivatives of taurine with fatty acids. Stability studies performed with the new SAM salts indicate that SAM becomes much more stable when it interacts with large size anions and in fact, more than 84% of the SAM is recovered after 36 months in lyophilized samples. The high stability of the new products widens the possibility of new therapeutic applications of SAM in human therapy.
Keywords: S-adenosyl-l-methionine; Sodium N-ole-1-oyltaurate; Stability; Liposoluble derivatives;

The main aim of this paper was to investigate the influence of temperature, pH and ionic interactions on the structural stability and surface adsorption of a recombinant factor VIII product, r-VIII SQ. The interaction of r-VIII SQ with glass and air interfaces, and possible means of increasing the stability of the formulation, were also investigated. The stability of r-VIII SQ was followed by measuring the biological activity (VIII:C), by circular dichroism (CD) studies and by the measurement of surface tension using the pendant drop method. The results show that the surface tension decreased exponentially with time; this decrease was more pronounced above 20°C, indicating increased conformational flexibility of the protein with increased temperatures. Far UV CD spectra were not influenced in the range 5–55°C and near UV CD measurements did not indicate structural changes below 45°C. During agitation at 25°C, VIII:C was lost rapidly in formulations without a macromolecular additive. Nonionic surfactants such as polysorbate 80 and polysorbate 20 protected VIII:C to an equally high degree against surface adsorption. Albumin was less effective, but it is possible that this is because it is a protein itself and may have been affected by the agitation. The addition of 300 mg/ml of sucrose improved the long term stability of VIII:C, a finding most likely explained by the theory of preferential hydration. Near UV CD spectra at acidic or basic pH mainly indicated changes around 242 nm, especially at low ionic strength. Addition of 10 mM EDTA at pH 7 resulted in similar changes. This effect was completely reversed by the addition of an excess of Ca2+, Sr2+ or Mg2+ ions. In conclusion, CD spectra and surface tension measurements of r-VIII SQ did not reveal any temperature-induced conformational changes in the temperature range 5–20°C; changes were first noted at elevated temperatures. Surface adsorption of r-VIII SQ during agitation was prevented by the addition of a nonionic surfactant. Preferential hydration improved the storage stability of the protein but did not directly prevent its surface adsorption. The structural integrity of the molecule was preserved at pH 7, at an increased ionic strength and in the presence of some divalent metal ions (Ca2+, Sr2+ or Mg2+).
Keywords: Protein stability; Factor VIII; Conformation; CD; Surface tension; Metal ions;

A self-emulsifying system (SES), a mixture of an oil and a surfactant which forms an oil-in-water emulsion, is expected to improve the in vitro drug dissolution and enhance the in vivo drug absorption. In this study, a poorly water-soluble drug, indomethacin (IDM) was incorporated into the SES to increase bioavailability. The SES with 30% of Tween 85 and 70% of ethyl oleate, EO (w/w) was selected as an optimized formulation (high drug loading, low surfactant concentration, and small particle size). After an oral administration of the SES containing IDM and IDM suspension, (IDM was suspended in methyl cellulose), 22.5 mg/kg as IDM, to rats, the area under the plasma concentration–time curve from time zero to the last measured time in plasma, 12 h (AUC0–12 h) was significantly greater (57% increase) in the SES, suggesting that oral absorption of IDM increased significantly by the SES. After a rectal administration of gelatin hollow type suppositories, filled with the SES containing IDM and IDM powder physically mixed with the SES, 22.5 mg/kg, to rats, the AUC0–12 h also increased significantly (41% increase) by the SES, suggesting that rectal absorption of IDM also increased significantly by the SES.
Keywords: Indomethacin; SES (Tween 85: ethyl oleate, 3:7); Gelatin hollow type rectal suppository; Rats;

Desolvation process and surface characterisation of protein nanoparticles by C Weber; C Coester; J Kreuter; K Langer (91-102).
The objective of the present study was to characterise and optimise the desolvation process of human serum albumin (HSA) for the preparation of nanoparticles and to characterise the resulting colloidal system. Following the desolvation of the protein, the resulting nanoparticles were stabilised by the addition of varying amounts of glutaraldehyde or by heat denaturation. The particle size, zeta potential, and the number of available amino groups on the surface of the nanoparticles were determined. The amino groups were quantified by a spectrophotometric method using 2,4,6-trinitrobenzenesulfonic acid (TNBS). The results indicated that the particle size depended mainly on the amount of desolvating agent added, but not on the amount of cross-linker or the kind of cross-linking procedure. Increasing amounts of glutaraldehyde reduced the number of amino groups on the surface of HSA nanoparticles and also decreased the zeta potential of the carrier system. The temperature and heat denaturation time only had an influence on the stability of the nanoparticles but not on the amount of amino groups or the particle size. It was shown that heat denatured HSA nanoparticles possessed the greatest number of amino groups on their surface. Additional experiments for the characterisation of gelatin A and B nanoparticles were performed.
Keywords: Nanoparticles; Human serum albumin (HSA); Gelatin; Surface characterisation; Desolvation procedure; Amino group determination;

Thermosetting microemulsions and mixed micellar solutions as drug delivery systems for periodontal anesthesia by Marie Scherlund; Martin Malmsten; Peter Holmqvist; Arne Brodin (103-116).
In the present study, thermosetting microemulsions and mixed micellar solutions were investigated as drug delivery systems for anesthetizing the periodontal pocket. The structure of the systems, consisting of the active ingredients lidocaine and prilocaine, as well as two block copolymers (Lutrol® F127 and Lutrol® F68), was investigated by NMR spectroscopy and photon correlation spectroscopy (PCS). The results obtained for dilute (1–3% w/w) solutions show discrete micelles with a diameter of 20–30 nm and a critical micellization temperature of 25–35°C. Gel permeation chromatography (GPC) was used to study the distribution of the active ingredients, and indicates a preferential solubilization of the active components in micelles over unimers. Analogous to the Lutrol® F127 single component system these formulations display an abrupt gelation on increasing temperature. The gelation temperature was found to depend on both the drug ionization and concentration. These systems have several advantages over emulsion-based formulations including good stability, ease of preparation, increased drug release rate, and improved handling due to the transparency of the formulations.
Keywords: Microemulsion; Lidocaine; Prilocaine; PEO-PPO-PEO block copolymers; Periodontal pocket; Temperature of gelation;

Sodium salts are often used in drug formulation but their partial solubility parameters are not available. Sodium alters the physical properties of the drug and the knowledge of these parameters would help to predict adhesion properties that cannot be estimated using the solubility parameters of the parent acid. This work tests the applicability of the modified extended Hansen method to determine partial solubility parameters of sodium salts of acidic drugs containing a single hydrogen bonding group (ibuprofen, sodium ibuprofen, benzoic acid and sodium benzoate). The method uses a regression analysis of the logarithm of the experimental mole fraction solubility of the drug against the partial solubility parameters of the solvents, using models with three and four parameters. The solubility of the drugs was determined in a set of solvents representative of several chemical classes, ranging from low to high solubility parameter values. The best results were obtained with the four parameter model for the acidic drugs and with the three parameter model for the sodium derivatives. The four parameter model includes both a Lewis-acid and a Lewis-base term. Since the Lewis acid properties of the sodium derivatives are blocked by sodium, the three parameter model is recommended for these kind of compounds. Comparison of the parameters obtained shows that sodium greatly changes the polar parameters whereas the dispersion parameter is not much affected. Consequently the total solubility parameters of the salts are larger than for the parent acids in good agreement with the larger hydrophilicity expected from the introduction of sodium. The results indicate that the modified extended Hansen method can be applied to determine the partial solubility parameters of acidic drugs and their sodium salts.
Keywords: Partial solubility parameters; Benzoic acid; Sodium benzoate; Ibuprofen; Sodium ibuprofen; Extended Hansen method;

The purpose of this study was to test the ability of topical formulations of finasteride and flutamide to re-enlarge hair follicles in male-pattern baldness. This was evaluated by an experimental model of human scalp skin graft transplanted onto SCID mice. A comparison was made between formulations containing finasteride and flutamide, and a vehicle formulation in terms of the mean hairs per graft, length, diameter of the shafts, and structures of the growth stages of the hair. Flutamide and finasteride had a significantly higher effect (P<0.05) than the placebo in all the tested parameters, but flutamide demonstrated more hair per graft and longer hair shafts than finasteride (P<0.05). The number of hairs per graft for flutamide and finasteride groups were 1.22±0.47 and 0.88±0.95 hairs/0.5 mm2 graft, respectively, versus 0.35±0.6 hairs/graft for vehicle-treated graft. Similarly, hair lengths for flutamide and finasteride were 5.82±0.50 and 4.50±0.32 mm, respectively, versus 2.83±0.18 mm for the vehicle-treated grafts. An in vitro diffusion study of flutamide gel using hairless mouse skin demonstrated the beneficial effect of the vehicle composition in comparison with a hydroalcoholic solution or a gel containing no penetration enhancer. It is therefore suggested that this topical composition containing flutamide or finasteride may effectively result in regression of male-pattern baldness.
Keywords: Androgenetic alopecia; Flutamide; Finasteride; Topical drug delivery; Skin permeation; Mice;