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Literature data relevant to the decision to allow a waiver of in vivo bioequiva- lence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing lamivudine as the only active pharmaceutical ingredient were reviewed. The solubility and permeability data of lamivudine as well as its therapeutic index, its pharmacokinetic proper- ties, data indicating excipient interactions, and reported BE/bioavailability (BA) studies were taken into consideration. Lamivudine is highly soluble, but its permeability characteristics are not well-defined. Reported BA values in adults ranged from 82% to 88%. Therefore, lamivu- dine is assigned to the biopharmaceutics classification system (BCS) class III, noting that its permeability characteristics are near the border of BCS class I. Lamivudine is not a narrow therapeutic index drug. Provided that (a) the test product contains only excipients present in lamivudine IR solid oral drug products approved in the International Conference on Harmoniza- tion or associated countries in usual amounts and (b) the test product as well as the comparator product fulfills the BCS dissolution criteria for very rapidly dissolving; a biowaiver can be rec- ommended for new lamivudine multisource IR products and major post-approval changes of marketed drug products. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:2054-2063, 2011

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release solid oral dosage forms containing mefloquine hydrochloride as the only active pharmaceutical ingredient (API) are reviewed. The solubility and permeability data of mefloquine hydrochloride as well as its therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability studies were taken into consideration. Mefloquine hydrochloride is not a highly soluble API. Since no data on permeability are available, it cannot be classified according to the Biopharmaceutics Classification System with certainty. Additionally, several studies in the literature failed to demonstrate BE of existing products. For these reasons, the biowaiver cannot be justified for the approval of new multisource drug products containing mefloquine hydrochloride. However, scale-up and postapproval changes (HHS-FDA SUPAC) levels 1 and 2 and most EU type I variations may be approvable without in vivo BE, using the dissolution tests described in these regulatory documents.

The aim of this study was to evaluate the ability of TMC, with different degrees of quaternization, to increase insulin absorption in vivo following nasal and rectal administration in rats. Two batches of TMC with different degrees of quaternization (TMC-L, 12.3% quaternized and TMC-H, 61.2% quaternized) and chitosan hydrochloride were administered intranasally (0.25 and 0.5% w/v) and rectally (0.5% w/v) with insulin (4 IU/kg body weight), at a pH of 4.40 and 7.40, in rats. Blood samples were taken over a period of 2 h for measurement of blood glucose levels and plasma insulin levels. Local toxicity evaluation was done by histological examination of the nasal and rectal epithelia. At pH 4.40 all these polymers were able to increase nasal and rectal insulin absorption, compared to the control groups. However, at a pH of 7.40, only TMC-H was able to increase the nasal and rectal absorption of insulin. These results relate to the insolubility of chitosan hydrochloride at neutral pH values, while the charge density of TMC-L is still too low for any significant interaction at pH 7.40. Histological evaluation of the nasal and rectal eptihelia shows no changes in the morphology of the cells after exposure to these polymers. Only slight congestion of the nasal submucosa was observed and all these polymers led to a mild increase in mucus secretion at pH 4.40. Highly quaternized TMC proves to be a potent absorption enhancer in vivo, especially at neutral pH values where chitosan salts are ineffective.

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing doxycycline hyclate are reviewed. According to the Biopharmaceutics Classification System (BCS), doxycycline hyclate can be assigned to BCS Class I. No problems with BE of IR doxycycline formulations containing different excipients and produced by different manufacturing methods have been reported and hence the risk of bio in equivalence caused by these factors appears to be low. Doxycycline has a wide therapeutic index. Further, BCS-based dissolution methods have been shown to be capable of identifying formulations which may dissolve too slowly to generate therapeutic levels. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing doxycycline hyclate as the single Active Pharmaceutical Ingredient (API) provided that (a) the test product contains only excipients present in doxycycline hyclate IR solid oral drug products approved in the International Conference on Harmonization (ICH) or associated countries; and (b) the comparator and the test products comply with the BCS criteria for “very rapidly dissolving” or, alternatively, when similarity of the dissolution profiles can be demonstrated and the two products are “rapidly dissolving.”. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1639–1653, 2010

The biopolymer chitosan, derived from chitin by deacetylation, has been considered for drug and vaccine carrier systems due to its biocom- patibility, biodegradability, and absence of toxicity. In addition, chitosan serves as the base for the synthesis of various derivatives, having, for example, increased aqueous solubility at a wide pH range or offering the opportunity for functionalization with targeting moieties. As the polymer was described as displaying adjuvant properties also, chitosan and its derivatives are used in vaccine delivery systems, alone or in combination with other polymers. This review highlights the current status of the use of chitosan and its derivatives in vaccine carrier design.

It has long been known that protection against pathogens invading the organism via mucosal surfaces correlates better with the presence of specific antibodies in local secretions than with serum antibodies. The most effective way to induce mucosal immunity is to administer antigens directly to the mucosal surface. The development of vaccines for mucosal application requires antigen delivery systems and immunopotentiators that efficiently facilitate the presentation of the antigen to the mucosal immune system. This review provides an overview of the events within mucosal tissues that lead to protective mucosal immune responses. The understanding of those biological mechanisms, together with knowledge of the technology of vaccines and adjuvants, provides guidance on important technical aspects of mucosal vaccine design. Not being exhaustive, this review also provides information related to modern adjuvants, including polymeric delivery systems and immunopotentiators.

During persistent infection and hypoxic-stress, Mycobacterium tuberculosis (Mtb) expresses a series of Mtb latency antigens. The aim of this study was to evaluate the immunogenicity of a DNA vaccine encoding the Mtb latency antigen Rv1733c and to explore the effect of pulmonary delivery and co-formulation with poly (d,l-lactide-co-glycolide) (PLGA)-polyethyleneimine (PEI) nanoparticles (np) on host immunity. Characterization studies indicated that PLGA-PEI np kept their nanometer size after concentration and were positively charged. The np were able to mature human dendritic cells and stimulated them to secrete IL-12 and TNF-alpha comparable to levels observed after lipopolysaccharide (LPS) stimulation. Mtb latency antigen Rv1733c DNA prime combined with Rv1733c protein boost enhanced T cell proliferation and IFN-gamma secretion in mice in response to Rv1733c and Mtb hypoxic lysate. Rv1733c DNA adsorbed to PLGA-PEI np and applied to the lungs increased T cell proliferation and IFN-gamma production more potently compared to the same vaccinations given intramuscularly. The strongest immunogenicity was obtained by pulmonary priming with np-adsorbed Rv1733c DNA followed by boosting with Rv1733c protein. These results confirm that PLGA-PEI np are an efficient DNA vaccine delivery system to enhance T cell responses through pulmonary delivery in a DNA prime/protein boost vaccine regimen.

Literature data are reviewed regarding the scientific advisability of allowing a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing either diclofenac potassium and diclofenac sodium. Within the biopharmaceutics classification system (BCS), diclofenac potassium and diclofenac sodium are each BCS class II active pharmaceutical ingredients (APIs). However, a biowaiver can be recommended for IR drug products of each salt form, due to their therapeutic use, therapeutic index, pharmacokinetic properties, potential for excipient interactions, and performance in reported BE/bioavailability (BA) studies, provided: (a) test and comparator contain the same diclofenac salt; (b) the dosage form of the test and comparator is identical; (c) the test product contains only excipients present in diclofenac drug products approved in ICH or associated countries in the same dosage form, for instance as presented in this paper; (d) test drug product and comparator dissolve 85% in 30 min or less in 900 mL buffer pH 6.8, using the paddle apparatus at 75 rpm or the basket apparatus at 100 rpm; and (e) test product and comparator show dissolution profile similarity in pH 1.2, 4.5, and 6.8.

The aim of this investigation was to design a novel Gas Empowered Drug Delivery (GEDD) system for CO 2 forced transport of peptide drugs together with mucoadhesive polymers to the surface of the small intestine. The GEDD effect of the core tablet was achieved using CO 2 gas to push insulin together with the mucoadhesive excipients poly(ethyleneoxide) (PEO) and the permeation enhancer trimethyl chitosan (TMC) to the surface of the small intestine. The in-vitro insulin release showed that almost 100% of the insulin was released from enterically coated tablets within 30 min at pH 6.8. The designed GEDD system was shown to increase the insulin transport by approximately 7 times in comparison with the free insulin across sheep's intestine ex-vivo. Three different peroral formulations were administered to male rabbits: F1 containing no TMC or PEO, F2 containing PEO but no TMC and F3 containing both PEO and TMC. The administrations of insulin using the formulation F1 resulted in a low FR value of 0.2% ± 0.1%, while the formulations F2 and F3 resulted in a much higher FR values of 0.6 ± 0.2% and 1.1% ± 0.4%, respectively. Hence, the insulin permeation achieved by the GEDD system is primarily due to the enhancing effect of TMC and the mucoadhesive properties of PEO both of which synergistically increase the bioavailability of insulin.

article i nfo We recently described a delivery system that is composed of a chitosan core to which the hepatitis B surface antigen (HBsAg) was adsorbed and subsequently coated with sodium alginate. In this present work, alginate coated chitosan nanoparticles were evaluated as a subcutaneous adjuvant for HBsAg. HBsAg loaded, alginate coated or uncoated chitosan nanoparticles, associated or not with CpGODN were subcutaneously administered to mice and several immunological parameters were evaluated. A high anti-HBsAg IgG titer (2271±120 mIU/ml), with the majority of antibodies being of Th2 type, was observed within group I, vaccinated with HBsAg loaded onto coated nanoparticles. However, regarding cellular immune response, no significant differences were observed for antigen-specifi cs plenocyte proliferation or for the secretion of IFN-γ and IL-4, when compared to the control group. The co-delivery of antigen-loaded nanoparticles in the presence of the immunopotentiator, CpG ODN 1826, resulted in an increase of anti-HBsAg IgG titers that was not statistically different from the first group; however, an increase of the IgG2a/IgG1 ratio from 0.1 to 1.0 and an increase (pb0.01) of the IFN-γ production by the splenocytes stimulated with the HBV antigen was observed. The enhancement of the immune response observed with the antigen-loaded nanoparticles demonstrated that chitosan is a promising platform for parenteral HBsAg delivery and, when co-administered with the CpG ODN, resulted in a mixed Th1/Th2 type immune response.

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing (biowaiver) for the approval of immediate release (IR) solid oral dosage forms containing aciclovir are reviewed. Aciclovir therapeutic use and therapeutic index, pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) studies were also taken into consideration in order to ascertain whether a biowaiver can be recommended. According to the Biopharmaceutics Classification System (BCS) and considering tablet strengths up to 400 mg, aciclovir would be BCS Class III. However, in some countries also 800 mg tablets are available which fall just within BCS Class IV. Aciclovir seems not to be critical with respect to a risk for bioinequivalence, as no examples of bioinequivalence have been identified. It has a wide therapeutic index and is not used for critical indications. Hence, if: (a) the test product contains only excipients present in aciclovir solid oral IR drug products approved in ICH or associated countries, for instance as presented in this article; and (b) the comparator and the test product both are very rapidly dissolving, a biowaiver for IR aciclovir solid oral drug products is considered justified for all tablet strengths.

Literature data are reviewed relevant to the decision for a biowaiver of immediate release (IR) solid oral dosage forms containing metoclopramide hydrochloride. In addition, new solubility data, obtained under Biopharmaceutics Classification System (BCS) conditions are presented. Metoclopramide HCl is conservatively assigned to BCS Class III. Taken also into consideration excipient interactions reported in metoclopramide drug products, its pharmacokinetic properties and therapeutic use and therapeutic index, a biowaiver can be recommended when: (a) the test product contains only excipients present also in metoclopramide HCl containing IR solid oral drug products approved in ICH or associated countries, for instance as presented in this paper, (b) in amounts in normal use in IR solid oral dosage forms, and (c) the test product and the comparator both comply with the criteria for very rapidly dissolving.

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing acetazolamide are reviewed. Acetazolamide's solubility and permeability characteristics according to the Biopharmaceutics Classification System (BCS), as well as its therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) problems are taken into consideration. The available data on solubility, on oral absorption and permeability are not sufficiently conclusive to classify acetazolamide with certainty. Taking a conservative approach, no biowaiver is considered justified for the registration of new multisource drug products. However, SUPAC level 1 and level 2 postapproval changes and most EU Type I variations can be approved waiving in vivo BE studies.

Insulin (INS), like other peptides, has low therapeutic activity when administered orally due to degradation by proteolytic enzymes. Polymeric nanoparticles have been introduced as a useful carrier for peptide oral delivery, because they can protect these compounds from degradation. The objective of the present study is to develop an INS nanoparticulate system by using chitosan (CS), triethylchitosan (TEC), and dimethyl-ethylchitosan (DMEC, a new quaternized derivative of CS). INS-polymer nanoparticles were prepared by the polyelectrolyte complexation method. The physicochemical properties of the nanoparticles including particle size distribution, zeta potential, and polydispersity index were determined by using dynamic light scattering technique. Transmission electron microscopy was also used to observe the morphology of the nanoparticles. The amount of INS loaded into the nanoparticles was determined by measuring the association efficiency and also the content of INS in the nanoparticles. In vitro release studies showed a relatively small burst effect at the beginning and then a sustained release characteristic for 5 hours.

Alginate coated chitosan nanoparticles were previously developed with the aim of protecting the antigen, adsorbed on the surface of those chitosan nanoparticles, from enzymatic degradation at mucosal surfaces. In this work, this new delivery system was loaded with the recombinant hepatitis B surface antigen (HBsAg) and applied to mice by the intranasal route. Adjuvant effect of the delivery system was studied by measuring anti-HBsAg IgG in serum, anti-HBsAg sIgA in faeces extracts or nasal and vaginal secretions and interferon-[gamma] production in supernatants of the spleen cells. The mice were primed with 10 [mu]g of the vaccine associated or not with nanoparticles and associated or not with 10 [mu]g CpG oligodeoxynucleotide (ODN) followed by two sequential boosts at three week intervals. The association of HBsAg with the alginate coated chitosan nanoparticles, administered intranasally to the mice, gave rise to the humoral mucosal immune response. Humoral systemic immune response was not induced by the HBsAg loaded nanoparticles alone. The generation of Th1-biased antigen-specific systemic antibodies, however, was observed when HBsAg loaded nanoparticles were applied together with a second adjuvant, the immunopotentiator, CpG ODN. Moreover, all intranasally vaccinated groups showed higher interferon-[gamma] production when compared to naïve mice. http://www.sciencedirect.com/science/article/B6T6C-4RR1NPN-2/1/beaa4e06ecb340a5a293ef1fd3b4c866

TMC and DEMC, quaternized derivatives of chitosan, have been shown to have penetration enhancement properties and able to open the tight junctions of the intestinal epithelia at neutral and alkaline pH environments. The use of the nanoparticulate systems has the advantage of protecting the peptidic drugs from the harsh environment of the gastrointestinal tract. Hence, the aim of this study was to synthesize and characterize TMC and DEMC, both with quaternization degrees of 50+/-5%, which were then used to prepare insulin nanoparticles with two different methods: ionotropic gelation and the polyelectrolyte complexation (PEC) techniques. The obtained nanoparticles were then characterized for size, zeta potential, insulin loading and release as well as antibacterial activities. The results showed that nanoparticles prepared by the PEC method had higher insulin loading efficiency and zeta potential than those made by the ionotropic gelation method and may subsequently be used for further in vitro, ex vivo and in vivo studies. Moreover, the antibacterial studies suggest that the polymers in free form have higher antibacterial activity against Gram-positive bacteria than in the nanoparticulate form.

Chitosan, a biodegradable and biocompatible polymer, has attracted great attention in the pharmaceutical and biomedical fields especially due to its properties to reversibly open the tight junctions of the epithelial tissues and to allow for paracellular transport of hydrophilic macromolecules. However, chitosan exhibits low solubility at pH values above 6 that may prevent its enhancing effects at the sites of intestinal absorption. Hence, a number of alkylated chitosan salts have been synthesized and characterized. These derivatives have been shown to have good solubility at neutral pH and act as effective permeation enhancers. In this study, two new derivatives of chitosan, C2—C6 trimethyl 6-amino-6-deoxy chitosan and C2—C6 triethyl 6-amino-6-deoxy chitosan were synthesized and characterized using 1H-NMR and FTIR spectra. Moreover, the zeta potential and the antibacterial properties of these polymers were compared to chitosan, trimethyl chitosan (TMC) and triethyl chitosan (TEC). Our results suggest that both C2—C6 trimethyl and triethyl 6-amino-6-deoxy chitosan, as highly water soluble polymers, have higher positive surface charge than chitosan, TMC, and TEC. Moreover, the new synthesized polymers show higher antibacterial activity against Gram-positive Staphylococcus aureus bacteria. Consequently, these polymers with substitution degrees of 50—60% may be good candidates for the enhancement of peptides in mucosal drug delivery.

Literature data relevant to the decision to allow a waiver of in vivo bioequi- valence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing ethambutol dihydrochloride as the only active pharmaceutical ingredient (API) are reviewed. Ethambutol dihydrochloride is a Biopharmaceutics Classification System (BCS) Class III drug with permeability properties approaching the border between BCS Class I and III. BE problems of ethambutol formulations containing different excipients and different dosages forms have not been reported and hence the risk of bioinequivalence caused by excipients is low. Ethambutol has a narrow therapeutic index related to ocular toxicity. However, as long as the prescribers' information of the test product stipulates the need for regular monitoring of ocular toxicity, the additional patient risk is deemed acceptable. It is concluded that a biowaiver can be recommended for IR solid oral dosage forms provided that the test product (a) contains only excipients present in ethambutol IR solid oral drug products approved in ICH or associated countries, for instance as presented in this paper, (b) complies with the criteria for ''very rapidly dissolving'' and (c) has a prescribers' information indicating theneed for testing thepatient's visionprior toinitiating ethambutol therapy and regularly during therapy. 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1350-1360, 2008

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing isoniazid as the only active pharmaceutical ingredient (API) are reviewed. Isoniazid's solubility and permeability characteristics according to the Biopharmaceutics Classification System (BCS), as well as its therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions and reported BE/bioavailability (BA) problems were taken into consideration. Isoniazid is “highly soluble” but data on its oral absorption and permeability are inconclusive, suggesting this API to be on the borderline of BCS Class I and III. For a number of excipients, an interaction with the permeability is extreme unlikely, but lactose and other deoxidizing saccharides can form condensation products with isoniazid, which may be less permeable than the free API. A biowaiver is recommended for IR solid oral drug products containing isoniazid as the sole API, provided that the test product meets the WHO requirements for “very rapidly dissolving” and contains only the excipients commonly used in isoniazid products, as listed in this article. Lactose and/or other deoxidizing saccharides containing formulations should be subjected to an in vivo BE study. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci

In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of a monovalent influenza subunit vaccine was investigated. The antigen-loaded nanoparticles were prepared by mixing a solution containing TMC and monovalent influenza A subunit H3N2 with a tripolyphosphate (TPP) solution, at ambient temperature and pH 7.4 while stirring. The nanoparticles had an average size of about 800 nm with a narrow size distribution and a positive surface charge. The nanoparticles showed a loading efficiency of 78% and a loading capacity of 13% (w/w). It was shown that more than 75% of the protein remained associated with the TMC nanoparticles upon incubation of the particles in PBS for 3 h. The molecular weight and antigenicity of the entrapped hemagglutinin was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. Single i.n. or i.m. immunization with antigen-loaded TMC nanoparticles resulted in strong hemagglutination inhibition and total IgG responses. These responses were significantly higher than those achieved after i.m. administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The i.n. administered antigen-TMC nanoparticles induced higher immune responses compared to the other i.n. antigen formulations, and these responses were enhanced by i.n. booster vaccinations. Moreover, among the tested formulations only i.n. administered antigen-containing TMC nanoparticles induced significant IgA levels in nasal washes of all mice. In conclusion, these findings demonstrate that TMC nanoparticles are a potent new delivery system for i.n. administered influenza antigens. (c) 2006 Elsevier Ltd. All rights reserved.

N, N-dimethyl N-ethyl chitosan (DMEC), a quanternized derivative of chitosan was synthesized based on a modified two-step method via a 22 factorial design to optimize the preparative conditions. The degree of deacetylation of the starting chitosan was determined by FTIR and NMR methods and was 95%. In the first step of the synthesis, mono-ethyl chitosan was prepared by introducing an ethyl group onto the amine group of chitosan via a Schiff base and in the next step methyl iodide was added to produce DMEC which was water soluble in a pH range of 4-8. The DMEC polymers with different degrees of quaternization were obtained and fully characterized using FTIR and 1H-NMR spectroscopic methods. Based on 1H-NMR calculations, the degree of quaternization was 52% by optimizing the two-step process.

The design of particulate vaccine delivery systems, particularly for mucosal surfaces, has been a focus of interest in recent years. In this context, we have previously described the development and the characterization of a new nanosized delivery system, consisting of a model antigen adsorbed to chitosan particles and coated with sodium alginate. In the present work the ovalbumin release profiles from these coated nanoparticles in different pH buffers were investigated and compared to those of the uncoated particles. Cytotoxicity of the polymers and nanoparticles was assessed using the MTT assay. Finally, particle uptake studies in rat Peyer's patches were performed. It was demonstrated that the coating of the nanoparticles with sodium alginate not only avoided a burst release observed with uncoated particles but also increased the stability of the particles at pH 6.8 and 7.4 at 37 °C. At neutral pH, the release was lower than 5% after 3.5 h incubation in a low ionic strength buffer. For both, chitosan and alginate polymers, and for the nanoparticles, comparable cell viability data close to 100%, were obtained. Additionally, based on confocal laser scanning microscopy observations, it was shown that alginate coated nanoparticles were able to be taken up by rat Peyer's patches, rendering them suitable carriers for intestinal mucosal vaccination. http://www.sciencedirect.com/science/article/B6T3D-4K6CHRP-1/1/a257a285900ecde6d4747e1df67e1a82

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release (IR) solid oral dosage forms containing cimetidine are reviewed. According to the current Biopharmaceutics Classification System (BCS), cimetidine would be assigned to Class III. Cimetidine's therapeutic use and therapeutic index, its pharmacokinetic properties, data related to the possibility of excipient interactions, and reported BE/bioavailability (BA) problems were also taken into consideration. On the basis of the overall evidence, a biowaiver can be recommended for cimetidine IR products, provided that the test product contains only those excipients reported in this paper in their usual amounts, and that the test and the comparator drug products both are “rapidly dissolving” as per BCS.

Literature data are reviewed on the properties of acetaminophen (paracetamol) related to the biopharmaceutics classification system (BCS). According to the current BCS criteria, acetaminophen is BCS Class III compound. Differences in composition seldom, if ever, have an effect on the extent of absorption. However, some studies show differences in rate of absorption between brands and formulations. In particular, sodium bicarbonate, present in some drug products, was reported to give an increase in the rate of absorption, probably caused by an effect on gastric emptying. In view of Marketing Authorizations (MAs) given in a number of countries to acetaminophen drug products with rapid onset of action, it is concluded that differences in rate of absorption were considered therapeutically not relevant by the Health Authorities. Moreover, in view of its therapeutic use, its wide therapeutic index and its uncomplicated pharmacokinetic properties, in vitro dissolution data collected according to the relevant Guidances can be safely used for declaring bioequivalence (BE) of two acetaminophen formulations. Therefore, accepting a biowaiver for immediate release (IR) acetaminophen solid oral drug products is considered scientifically justified, if the test product contains only those excipients reported in this paper in their usual amounts and the test product is rapidly dissolving, as well as the test product fulfils the criterion of similarity of dissolution profiles to the reference product.

Literature data are reviewed on the properties of ibuprofen related to the biopharmaceutics classification system (BCS). Ibuprofen was assessed to be a BCS class II drug. Differences in composition and/or manufacturing procedures were reported to have an effect on the rate, but not the extent of absorption; such differences are likely to be detectable by comparative in vitro dissolution tests. Also in view of its therapeutic use, its wide therapeutic index and uncomplicated pharmacokinetic properties, a biowaiver for immediate release (IR) ibuprofen solid oral drug products is scientifically justified, provided that the test product contains only those excipients reported in this paper in their usual amounts, the dosage form is rapidly dissolving (85% in 30 min or less) in buffer pH 6.8 and the test product also exhibits similar dissolution profiles to the reference product in buffer pH 1.2, 4.5, and 6.8.

In this report we present in detail a non-invasive pulmonary application method that can be a useful tool in studying drug and vaccine delivery to the lower airways. In this method the formulation is sprayed directly into the lungs of mice via the endotracheal route using a MicroSprayer aerolizer. Mean droplet size produced was 8 microm, appropriate for deposition in the large airways. Endotracheal application of suspension of fluorescent nanospheres, 200 nm in size, by this method resulted in nanoparticle deposition in the smaller airways (bronchi and bronchioles). Mice showed full recovery one day after administration of 50 microl of formulation. Furthermore, no mortality was observed as a result of the technique. We conclude that this endotracheal application is a useful tool for studying pulmonary drug delivery in mice. The technique is especially useful for the pulmonary application of vaccines, since it enables multiple administrations without a need for analgesics.

Literature data on the properties of chloroquine phosphate, chloroquine sulfate, and chloroquine hydrochloride related to the Biopharmaceutics Classification System (BCS) are reviewed. The available information indicates that these chloroquine salts can be classified as highly soluble and highly permeable, i.e., BCS class I. The qualitative composition of immediate release (IR) tablets containing these Active Pharmaceutical Ingredients (APIs) with a Marketing Authorization (MA) in Belgium (BE), Germany (DE), Finland (FI), and The Netherlands (NL) is provided. In view of these MA's and the critical therapeutic indication of chloroquine, it is assumed that the registration authorities had evidence that these formulations are bioequivalent to the innovator. It is concluded that IR tablets formulated with these excipients are candidates for a biowaiver.

Mucosal immunity establishes the first line of defence against pathogens entering the body via mucosal surfaces. Besides eliciting both local and systemic immunity, mucosal vaccination strategies that are non-invasive in nature may increase patient compliance and reduce the need for vaccine application by trained personnel. A relatively new concept is mucosal immunization using DNA vaccines. The advantages of DNA vaccines, such as the opportunity to combine the genetic information of various antigen epitopes and stimulatory cytokines, the enhanced stability and ease of production make this class of vaccines attractive and suitable for mucosal application. In contrast to the area of intranasal vaccination, only a few recent studies have focused on pulmonary immunization and the involvement of the pulmonary immune system in eliciting protective immune responses against inhaled pathogens. This review focuses on DNA vaccine delivery to the lung as a promising approach to prevent pulmonary-associated diseases caused by inhaled pathogens. Attractive immunological features of the lung as a site for immunization, the mechanisms of action of DNA vaccines and the pulmonary application of such vaccines using novel delivery systems will be discussed. We also examine pulmonary diseases prone to prevention or therapeutical intervention by application of DNA vaccines.

Pulmonary immunization against inhaled pathogens such as Mycobacterium tuberculosis would induce local and systemic immune responses and protect from entry and dissemination of the pathogen. The aim of this study was to evaluate cationic submicron emulsion as a potential carrier for DNA vaccines to the lung. DNA loaded emulsions were 128-152 nm in size and retained positive zeta potential above +40 mV during 3 months of storage. Loading efficiency was above 99%, DNA was protected from DNase I degradation up to 60 min and was stable in presence of 75% fetal calf serum (FCS). The plasmid DNA was detected in the endo-lysosomal compartment of the human bronchial cell line, Calu-3, 6 h after application. No cytotoxic effect on these cells was observed. Human dendritic cells were matured in presence of DNA loaded emulsion, although to a lesser extent than DNA solution indicating slower release and lower exposure to unmethylated CpG sequences. These results indicate that cationic submicron emulsions are potential DNA vaccine carriers to the lung since they are able to transfect pulmonary epithelial cells, which possibly induce cross priming of antigen presenting cells and directly activate dendritic cells, resulting in stimulation of antigen specific T-cells.

In recent years, many complex oral drug delivery systems have been developed using various polymers in order to achieve better drug targeting and drug absorption in the intestinal tract. Superporous hydrogel (SPH) and SPH composite (SPHC)-based drug delivery systems were also developed for the targeted delivery of peptide drugs into the intestinal tract. In the present study, the retention time of SPHC polymer is studied in man using the scintigraphy technique. To that purpose, SPHC polymers were radiolabelled with Tc-99m and administered orally in an enteric-coated gelatin capsule. The location of the radiolabelled polymer was monitored in five healthy volunteers while the subjects were sitting in front of a large field of view gamma camera. The results showed that enteric-coated gelatin capsules remained in the stomach for 75 to 150 min after oral administration to fasted volunteers and that the SPHC polymers thereafter attached to the upper part of the small intestine for at least 45 to 60 min due to their mechanical fixation properties. No discomfort was observed in any of the volunteers after oral administration of these polymers, which indicates that they are safe to be applied for oral drug delivery systems in man.

N-Trimethyl chitosan chloride (TMC) is a soluble chitosan derivative that shows effective enhancing properties for peptide and protein drug transport across mucosal membranes. TMC was synthesized by reductive methylation of chitosan in an alkaline environment at elevated temperature. The number of methylation process steps and the base used in the process was demonstrated to affect the degree of quaternization of the primary amino group and methylation of 3- and 6-hydroxyl groups. 1H-Nuclear magnetic resonance spectra showed that the degree of quaternization of TMC was higher when using sodium hydroxide as the base compared to using dimethyl amino pyridine. The degrees of quaternization as well as O-methylation of TMC increased with the number of reaction steps. O-Methylation resulted in decreased solubility of TMC. The high degree of quaternization of TMC with a low degree of O-methylation was prepared by employing one reaction step with two subsequent addition steps and a controlled alkaline environment of the mixture reaction.

The aim of this study was to investigate and modify the potential of drug-delivery systems based on superporous hydrogel (SPH) for improving the intestinal transport of the peptide drug desmopressin in vitro. The swelling properties and mechanical strength of SPHs were studied. The release profile of desmopressin was investigated by changing the composition of excipients in the formulations. Subsequently, the ability of the SPH-based drug-delivery systems to enhance the transport of desmopressin across porcine intestine was performed in vitro. The swelling properties and mechanical strength of SPHs were affected by the addition of the disintregrant AcDiSol ® . This disintregrant reduced the swelling ratio to 10% and the time to 80% swelling was retarded by 3–5 min in comparison to the negative control. AcDiSol ® increased the mechanical strength, according to the increasing of penetration pressure value, the pressure that the punch can penetrate the gel, of the SPHs. The transport of desmopressin across the intestinal mucosa in vitro was enhanced four- and six-fold by applying SPH, with AcDiSol ® , in the absence and presence of the additional absorption enhancer trimethyl chitosan chloride, respectively, in comparison to the negative control. It is concluded that drug-delivery systems based on SPHs are promising for enhancing the intestinal absorption of desmopressin.

In this paper we evaluated chitosan microparticles as a vaccine delivery system as well as the mucosal adjuvant LTK63, a nontoxic Escherichia coli enterotoxin (LT) mutant for the intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice receiving intranasally the CRM-MenC vaccine formulated with chitosan microparticles and the LTK63 mutant showed higher titers of systemic and mucosal antibodies specific for the group C meningococcal polysaccharide as compared to those receiving the vaccine subcutaneously. In addition, high bactericidal activity was found in serum samples of mice immunized intranasally with the conjugated vaccine formulated together with the microparticles and the LT mutant. These results demonstrate that the concomitant use of chitosan microparticles and the LTK63 mutant significantly enhances the immunogenicity and the protective efficacy of vaccines given intranasally.

The major disadvantage concerning clinical use of bishosphonate drugs, like clodronate, is their poor and variable absorption after oral administration. The objective of this study was to assess the effects of four different absorption enhancers—palmitoyl carnitine chloride (PCC), N -trimethyl chitosan chloride (TMC), sodium caprate (C10), and ethylene glycol-bis(β-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid (EGTA)—on the transport of clodronate using Caco-2 cell culture model. The transport experiments were performed in a normal (1.3 mM) and in a minimum-calcium concentration (apically calcium-free medium and basolaterally 100 μM calcium concentration). In the normal calcium concentration, a strong enhancement in clodronate permeation was observed with the enhancers: EGTA (2.5 mM), TMC (1.5% w/v), and PCC (0.2 mM) increased the transport of 1 mM clodronate 190-, 20-, and 10-fold, respectively, and the transport of 10 mM clodronate 130-, 70-, and 35-fold. In the minimum-calcium concentration, the effects of the absorption enhancers on the transport of clodronate were not so potent: TMC, PCC, and EGTA caused 2- to 20-fold enhancement in clodronate permeation whereas C10 (10 mM) was without any effect. According to the results, the permeation of clodronate through Caco-2 cells could be significantly promoted by the absorption enhancers, which cause widening of the tight junctions and, thus, increase the permeability of the paracellular route.

In this study, the ability of chitosan microparticles to enhance both the systemic and local immune responses against diphtheria toxoid (DT) after oral and nasal administration in mice was investigated.Firstly, DT was associated to chitosan microparticles to determine antigen loading and release. Then DT loaded chitosan microparticles, DT in phosphate buffered saline (PBS) and empty chitosan microparticles (as controls) were fed intragastrically and administered nasally to mice. Mice were also subcutaneously immunised with DT associated with alum. All mice were vaccinated in week 1 and boosted in week 3. Sera were analysed for anti-DT IgG and nasal washings and faeces for anti-DT IgA titres using an enzyme linked immunosorbent assay. Loading capacities of about 25% and loading efficacies of about 100% were obtained after loading the chitosan microparticles with DT. No DT was released at 37 degrees C in PBS. Compared to intragastrical feeding with DT in PBS, a strong enhancement of the systemic and local immune responses against DT were found in mice fed with DT loaded chitosan microparticles. In addition, a dose-dependent immune reaction was observed for mice vaccinated with different doses of DT associated to chitosan microparticles. Significant systemic humoral immune responses were also found after nasal vaccination with DT associated to chitosan microparticles.DT associated to chitosan microparticles results in protective systemic and local immune response against DT after oral vaccination, and in significant enhancement of IgG production after nasal administration. Hence, these in vivo experiments demonstrate that chitosan microparticles are very promising mucosal vaccine delivery systems.

Peat and various peat extracts have been successfully applied for a variety of clinical indications. Quite apart from the physico-thermal effects, new studies point towards the so-called "chemical effects" of peat containing substances. These effects include a stimulatory response of the spontaneous contractile activity (SCA) of smooth muscle (SM) tissue. The effects are, however, dependent on the possible permeability of pharmacologically active substances as naturally occurring ingredients of peat. Since peat is a mixture of various products it is necessary to examine the various peat types based upon their biological activity on SM tissue. In order to unequivocally prove the pharmacological activity of cutaneous peat treatment, in vitro permeation measurements of these actives across excised human skin can be used.HPLC analysis revealed that aqueous peat extracts contain up to 18 fractions of water-soluble compounds of fulvic and ulmic acids. These compounds have been found to have a stimulatory response on the contractile activity of SM tissue. In vitro diffusion studies showed that the permeability of these substances across human full thickness skin (thickness: 200 um(-1)) is highly selective and the resulting stimulatory activity is dependent on the permeated fraction. Especially, the HPLC fractions 7-11 and 14 are able to permeate human skin. Fractions 7-11 show a moderate stimulatory effect of SCA on SM for more than 90 min whereas fraction 14 shows the strongest stimulatory effect which was, however, suppressed after 87 min. These results show that the cutaneous therapy with peat treatment results in transcutaneaous permeation of biologically active fulvic and ulmic acid derivatives explaining the additional "chemical" effect of peat treatment in clinical practice.

In this in vivo study, novel delivery systems based on superporous hydrogel (SPH) and SPH composite (SPHC) polymers were used to improve the intestinal absorption of insulin in healthy pigs. Six female pigs of approximately 35 kg body weight were used. A cannula was inserted into the jugular vein for blood sampling and a silicone fistula in the duodenum for administration of gelatin capsules containing the delivery systems or insulin solutions. The delivery systems consisted of two components, (1) conveyor system made of SPH and SPHC; (2) core containing insulin. The core was inserted either into the conveyor system (core inside, c.i.) or attached to the surface of conveyor system (core outside, c.o.). The following intestinal formulations were investigated: c.i., c.o. and intraduodenal (i.d.) administration of insulin solutions. Subcutaneous (s.c.) injection of insulin was also investigated for reasons of comparison. Blood samples were taken and analyzed for insulin and glucose concentrations. Relative bioavalibility values of 1.3+/-0.4 and 1.9+/-0.7% were achieved for c.o. and c.i. administrations, respectively. The bioavalibility for i.d. administration of insulin solution was 0.5+/-0.2%. These results indicate that the absorption of insulin was slightly increased using SPH/SPHC-based delivery systems. Furthermore, a large variability was observed, probably due to physiological and metabolic changes during the experiments. Blood glucose levels were slightly decreased after the c.o. and c.i administrations, whereas these levels did not decrease after i.d. administration of insulin solutions. In conclusion, SPH/SPHC-based delivery systems are able to enhance the intestinal absorption of insulin and are, therefore, considered as promising systems for peroral peptide drug delivery. However, insulin delivery from these delivery systems under in vivo have to be improved.

Calu-3 cells grown on microporous filters at an air interface for 16-18 days were incubated with the glucocorticosteroid (GCS) budesonide (BUD). Apparent permeability (P(app)) of BUD across Calu-3 cell monolayers was determined. Amount of BUD transported across Calu-3 cells was clearly concentration dependent, and no polarised transport was detected. When cells were loaded with BUD by incubation with drug solution (30 microM) for 2 h, 9.35 +/- 0.53% of the initial dose of BUD, corresponding to 5.5 microg BUD/mg cell protein was retained intracellularly, and released over a time period of 10 h. Apical release of BUD was significantly higher than release to the basolateral side of the monolayer. In comparison, when Calu-3 cells were loaded with fluticasone propionate (FP) solution (0.8 microM), about 20% of the initial dose of FP, corresponding to 0.3 microg FP/mg total cell protein content was detected intracellularly and released immediately (45 min). There was no difference in FP release between the apical and basolateral side of the cell layer. Mass spectrometry of cell extracts indicated the presence of fatty acid conjugates of BUD. We conclude that Calu-3 cells are able to store BUD by intracellular conjugation to fatty acids. We, therefore, suggest the use of the Calu-3 cell model as a tool for examination of local pharmacokinetics and metabolism of GCS at the bronchial epithelium.

Quaternized modifications of chitosan present characteristics that might be useful in DNA condensing and efficient gene delivery. Trimethylated chitosan (TMO) was synthesized from oligomeric chitosan (520 monomer units).TMOs spontaneously formed complexes (chitoplexes) with RSV-a3 luciferase plasmid DNA.These complexes were characterized by photon correlation spectroscopy and were investigated for their ability to transfect COS-1 and Caco-2 cell lines in the presence and absence of fetal calf serum and compared with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulphate) lipoplexes.Additionally, their effect on the viability of the respective cell cultures was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay.Results showed that quaternized chitosan oligomers were able to condense DNA and form complexes with a size ranging from 200 to 500 nm.Chitoplexes proved to transfect COS-1 cells, however, to a lesser extent than DOTAP–DNA lipoplexes.The quaternized oligomer derivatives appeared to be superior to oligomeric chitosan.The presence of fetal calf serum (FCS) did not affect the transfection efficiency of the chitoplexes, whereas the transfection efficiency of DOTAP– DNA complexes was decreased.Cells remained 100% viable in the presence of chitosan oligomers whereas viability of DOTAP treated cells decreased to � 50% in both cell lines.Both DOTAP–DNA lipoplexes and chitoplexes resulted in less transfection efficiency in Caco-2 cell cultures than in COS-1 cells; however quaternized chitosan oligomers proved to be superior to DOTAP. Effects on the viability of Caco-2 cells were similar to the effects observed in COS-1 cells.We conclude that trimethylated chitosan– DNA complexes present suitable characteristics and the potential to be used as gene delivery vectors. # 2001 Elsevier Science Ltd. All rights reserved.

Purpose. The aim of this study was to investigate the effect of elastic and rigid vesicles on the penetration of pergolide across human skin. Methods. Vesicles used consisted of the bilayer-forming surfactant L-595 (sucrose laurate ester) and the micelle-forming surfactant PEG-8-L (octaoxyethylene laurate ester), together with the stabilizer sulfosuccinate. A series of L-595/PEG-8-L/sulfosuccinate vesicles were investigated, ranging from very rigid to very elastic. Pergolide-loaded elastic and rigid vesicles were visualized using Cryo-TEM and characterized for size and stability. Transdermal penetration of pergolide from different vesicle compositions was studied in vitro using flow-through Franz diffusion cells. A saturated buffer solution served as the control. Results. Vesicle composition had a major effect on the physico-chemical characteristics, morphology and drug solubility of the vesicular system. L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles gave the best balance between vesicle stability and elasticity, as well as the highest drug solubility. Transport studies clearly showed that elastic vesicles were superior to rigid vesicles. Elastic vesicles enhanced the drug transport compared to the buffer control, although rigid vesicles decreased the drug transport. The best drug transport was achieved from L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles, resulting in a steady-state flux of 13.6 ± 2.3 ng/(h*cm2). This was a 6.2-fold increase compared to the most rigid vesicles. Conclusions. This study supports the hypothesis that elastic vesicles are superior to rigid vesicles as vehicles for transdermal drug delivery.

Purpose. The aim of this study was to investigate the enhancement of peroral octreotide absorption using delivery systems based on superporous hydrogel (SPH) and SPH composite (SPHC) polymers.

1. Transepithelial transport of flunisolide was studied in reconstituted cell monolayers of Calu-3, LLC-PK1 and the MDR1-P-glycoprotein transfected LLC-MDR1 cells. 2. Flunisolide transport was polarized in the apical (ap) to basolateral (bl) direction in Calu-3 cells and was demonstrated to be ATP-dependent. In LLC-MDR1 cells, flunisolide was transported in the bl to ap direction and showed no polarization in LLC-PK1 cells. 3. Non-specific inhibition of cellular metabolism at low temperature (4 degrees C) or by 2-deoxy-D-glucose (2-d-glu) and sodium azide (NaN(3)) abolished the polarized transport. Polarized flunisolide transport was also inhibited by the specific Pgp inhibitors verapamil, SDZ PSC 833 and LY335979. 4. Under all experimental conditions and in the presence of all used inhibitors, no decrease in the TransEpithelial Electrical Resistance (TEER) values was detected. From all inhibitors used, only the general metabolism inhibitors 2-deoxy-D-glucose and NaN(3), decreased the survival of Calu-3 cells. 5. Western blotting analysis and confocal laser scanning microscopy demonstrated the presence of MDR1-Pgp at mainly the basolateral side of the plasma membrane in Calu-3 cells and at the apical side in LLC-MDR1 cells. Mass spectroscopy studies demonstrated that flunisolide is transported unmetabolized across Calu-3 cells. 6. In conclusion, these results show that the active ap to bl transport of flunisolide across Calu-3 cells is facilitated by MDR1-Pgp located in the basolateral plasma membrane.

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyer's patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.

Chitosan is a non-toxic, biocompatible polymer that has found a number of applications in drug delivery including that of absorption enhancer of hydrophilic macromolecular drugs. Chitosan, when protonated (pH

Numerous studies have demonstrated that chitosan and their derivatives (N-trimethyl chitosan, mono-N-carboxymethyl chitosan) are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophylic macromolecules such as peptide and protein drugs and heparins. This absorption enhancing effect of chitosans is caused by opening of the intercellular tight junctions, thereby favouring the paracellular transport of macromolecular drugs. Chitosan nano- and microparticles are also suitable for controlled drug release. Association of vaccines to some of these particulate systems has shown to enhance the antigen uptake by mucosal lymphoid tissues, thereby inducing strong systemtic and mucosal immune responses against the antigens. The aspecific adjuvant activity of chitosans seems to be dependent on the degree of deacetylation and the type of formulation. From the studies reviewed it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery.

Novel drug delivery systems were developed for peroral administration of peptide and protein drugs for site specific mechanical fixation at the gut wall and with specific release patterns. These so-called shuttle systems were designed by using superporous hydrogels (SPH) and SPH composite (SPHC) as the conveyor of a core which contained the model compound N-α-benzoyl- l -arginine ethylester (BAEE). Two different types of shuttle systems were evaluated: (a) core inside the shuttle system, and (b) core attached to the surface of shuttle system. Each of these systems was made of two parts: (1) the conveyor system made of SPHC which is used for keeping the dosage form at specific site(s) of the GI tract by mechanical interaction of the dosage form with the intestinal membranes, and (2) the core containing the active ingredient and incorporated in the conveyor system. The effect of formulation composition of the core on the release profile of BAEE was investigated by changing the type and amount of excipients in the formulations. In addition, the effect of various enteric-coat layers on the release profile and dissolving of the dosage form was investigated. The systems were also characterized for trypsin inactivation and Ca2+ binding. The release profile of BAEE from the core formulation consisting of PEG 6000 microparticles or small tablets showed the desired burst release. When these core formulations were incorporated into the conveyor system made of SPH and SPHC, a suitable time-controlled release profile was obtained. Changing the type, concentration and thickness of the enteric-coat layer influenced the starting time of BAEE release from the dosage form, which indicates the necessary lag time for dissolving of the dosage form at any desired specific site of drug absorption in the intestine. Both SPH and SPHC were found to partly inhibit the activity of trypsin, due to two mechanisms: Ca2+ binding and entrapment of the enzyme in these polymers. In conclusion, the presently developed delivery systems demonstrate suitable in vitro characteristics with an appropriate time-controlled release profile, making these systems very promising for effective peroral delivery of peptide and protein drugs.