Transport,Uptake, and Metabolism of the Bis(pivaloyloxymethyl)-Ester Prodrug of 9-(2- Phosphonylmethoxyethyl)Adenine in an In Vitro Cell Culture System of the Intestinal Mucosa (Caco-2)

Purpose. To evaluate intestinal transport, uptake and metabolism characteristics of the bis(pivaloyloxymethyl)-ester [bis(POM)-ester] of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA]. Methods. Intestinal transport, uptake and metabolism of bis(POM)-PMEA were studied using an in vitro cell culture system of the intestinal mucosa (Caco-2 monolayers). Concentrations of bis(POM)-PMEA and its metabolites mono(POM)-PMEA and PMEA were determined using a reversed-phase HPLC method. Enzymatic stability of bis(POM)-PMEA was evaluated by incubation with purified liver carboxylesterase, homogenates of Caco-2 cells and scraped pig small intestinal mucosa. Results. The use of bis(POM)-PMEA as a prodrug of PMEA resulted in a significant increase in transport of total PMEA [bis(POM)-PMEA, mono(POM)-PMEA and PMEA] across Caco-2 monolayers. While transepithelial transport of PMEA (500 M) was lower than 0.1% during a 3 hr incubation period, transport of total PMEA after addition of bis(POM)-PMEA (100 M) amounted to 8.8% over the same incubation period. Only 23% of the amount transported appeared as intact bis-ester at the basolateral side, while 33% of this amount was free PMEA and 44% was mono(POM)-PMEA, suggesting susceptibility of the prodrug to chemical and enzymatic degradation. Uptake studies revealed that only negligible amounts of bis(POM)-PMEA (< 0.2%) were present inside the cells. Very high intracellular concentrations of PMEA were found 1.2 mM, after a 3 hr incubation with 50 M bis(POM)-PMEA), which suggests that PMEA was trapped inside the cells probably due to its negative charge. This explains that efflux of PMEA was relatively slow (25% of the intracellular amount in 3 hr). Enzymatic degradation of the prodrug by carboxylesterase was confirmed by incubation of bis(POM)-PMEA with purified enzyme (K_m = 87 M and V_max = 9.5 M/min). Incubation of bis(POM)-PMEA (10 M) with cell homogenate of Caco-2 monolayers and pig small intestinal mucosa produced similar degradation profiles. Conclusions. The use of the bis(POM)-prodrug significantly enhances the intestinal permeability of PMEA. Intracellular trapping of PMEA in the intestinal mucosa may result in slow release of PMEA to the circulation after oral administration of bis(POM)-PMEA.     

Llama antibody fragments have good potential for application as HIV type 1 topical microbicides

There is an urgent global need for preventive strategies against HIV-1 infections. Llama heavy-chain antibody fragments (VHH) are a class of molecules recently described as potent cross-clade HIV-1 entry inhibitors. We studied the potential of a VHH-based microbicide in an application-oriented fashion. We show that VHH can be inexpensively produced in high amounts in the GRAS organism Saccharomyces cerevisiae, resulting in a very pure and endotoxin free product. VHH are very stable under conditions they might encounter during transport, storage, or use by women. We developed active formulations of VHH in aqueous gel and compressed and lyophilized tablets for controlled release from an intravaginal device. The release profile of the VHH from, e.g., a vaginal ring suggests sufficient bioavailability and protective concentration of the molecule at the mucosal site at the moment of the infection. The ex vivo penetration kinetics through human tissues show that the VHH diffuse into the mucosal layer and open the possibility to create a second defense layer either by blocking the HIV receptor binding sites or by blocking the receptors of immune cells in the mucosa. In conclusion, our data show that VHH have a high potential for HIV-1 microbicide application because of their low production costs, their high stability, and their favorable release and tissue penetration properties.     

Assessment of Passive Intestinal Permeability Using an Artificial Membrane Insert System

Despite reasonable predictive power of current cell-based and cell-free absorption models for the assessment of intestinal drug permeability, high costs and lengthy preparation steps hamper their use. The use of a simple artificial membrane (without any lipids present) as intestinal barrier substitute would overcome these hurdles. In the present study, a set of 14 poorly water-soluble drugs, dissolved in 2 different media (fasted state simulated/human intestinal fluids [FaSSIF/FaHIF]), were applied to the donor compartment of an artificial membrane insert system (AMI-system) containing a regenerated cellulose membrane. Furthermore, to investigate the predictive capacity of the AMI-system as substitute for the well-established Caco-2 system to assess intestinal permeability, the same set of 14 drugs dissolved in FaHIF were applied to the donor compartment of a Caco-2 system. For 14 drugs, covering a broad range of physicochemical parameters, a reasonable correlation between both absorption systems was observed, characterized by a Pearson correlation coefficient r of 0.95 (FaHIF). Using the AMI-system, an excellent predictive capacity of FaSSIF as surrogate medium for FaHIF was demonstrated (r = 0.96). Based on the acquired data, the AMI-system appears to be a time- and cost-effective tool for the early-stage estimation of passive intestinal permeability for poorly water-soluble drugs.     

Drying of crystalline drug nanosuspensions-The importance of surface hydrophobicity on dissolution behavior upon redispersion

d-alpha-Tocopherol polyethylene glycol 1000 succinate (TPGS)-stabilized nanosuspensions (25wt%, relative to the drug weight) were produced by media milling for 9 model drug compounds [cinnarizine, griseofulvin, indomethacin, itraconazole, loviride, mebendazole, naproxen, phenylbutazone and phenytoin]. After 3 months of storage at room temperature, Ostwald ripening occurred in all of the samples, except for indomethacin. Whereas lowering the temperature could slow down the ripening, it markedly increased upon storage at 40 degrees C. As for ripening, settling generally became more pronounced at 40 degrees C compared to 4 degrees C. As the nanosuspensions were afflicted by Ostwald ripening and settling, we explored nanosuspension drying as a strategy to circumvent these stability issues. Spray-drying and freeze-drying were evaluated for nanosuspensions and coarse reference suspensions of the compounds. Nanoparticle agglomeration could be visually observed in all of the powders. To evaluate the effect of agglomeration on the key characteristic of drug nanocrystals (i.e. rapid dissolution), dissolution experiments were performed under poor sink conditions. It was found that the compounds could be categorized into 3 groups: (i) compounds for which it was impossible to differentiate between coarse and nanosized products (griseofulvin, mebendazole, naproxen), (ii) compounds that gave clear differences in dissolution profiles between the nanosized and the coarse products, but for which drying of the nanosuspensions did not decrease the dissolution performance of the product (indomethacin, loviride, phenytoin) and (iii) compounds that showed differences between coarse and nanosized products, but for which drying of the nanosuspensions resulted in a significant decrease of the dissolution rate (cinnarizine, itraconazole, phenylbutazone). To gain insight on the influence of the drug compound characteristics on the dissolution of the dried products, the dissolution behavior of the compounds of the second and the third group was linked to the compound's characteristics. It was found that compounds with a more hydrophobic surface resulted in agglomerates which were harder to disintegrate, for which dissolution was compromised upon drying. The same was found for compounds having higher logP values.     

Safety-assessment of 3-methoxyquercetin as an antirhinoviral compound for nasal application: effect on ciliary beat frequency

It has been shown that 5,7,3',4'-tetrahydroxy-3-O-methylflavone (3-MQ) exhibits antipicornaviral activity. In order to explore the potential of 3-MQ as an antirhinoviral compound for nasal application, the effect of 3-MQ on the ciliary beat frequency (CBF) of human nasal epithelial cells was studied in vitro in the absence or presence of solubility/absorption enhancers (hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or polysorbate 80). Nasal epithelial cells were obtained by protease digestion of surgical specimens of human nasal polyps, and used at confluency. The effect of 3-MQ (2, 10, and 20 microg/ml), HP-beta-CD (1, 3, and 10% (w/v)), polysorbate 80 (0.1, 0.3, and 1% (w/v)), and of the combination of 3-MQ with 3% HP-beta-CD or 1% polysorbate 80, on the CBF was determined by computerized microscope photometry 15 min after incubation with the test compounds; recovery was determined 35 min after rinsing. HP-beta-CD at 1 and 3% did not affect CBF; a reversible decrease (by 37%) was observed at 10%. Polysorbate 80 caused a reversible cilio-inhibitory effect of 40, 53, and 49% at 0.1, 0.3, and 1%, respectively. At 2 and 10 microg/ml, 3-MQ showed a reversible cilio-stimulatory effect of 18 and 14%, respectively. Combined with 3% HP-beta-CD, the reversible cilio-stimulatory effect of 2 microg/ml 3-MQ was preserved, while 10 and 20 microg/ml 3-MQ did not affect the CBF. The combination of polysorbate 80 (1%) and 3-MQ decreased the CBF, which could be attributed to the presence of polysorbate 80. In conclusion, no ciliotoxic effect could be observed for 3-MQ (up to 20 microg/ml) in the absence or presence of HP-beta-CD (3%). The potential of this combination as an antirhinoviral formulation for nasal application will be further explored.     

P-Glycoprotein Attenuating Effect of Human Intestinal Fluid

Purpose. To evaluate the effect of human intestinal fluid (HIF) on P-glycoprotein (P-gp)-mediated efflux. Methods. HIF was obtained from eight healthy volunteers by duodenal aspiration. HIF was applied at different concentrations (0-75%) to the apical compartment of the Caco-2 system. Cyclosporin A (CsA) was used as a model compound for P-gp mediated efflux. Results. When the bidirectional transport of CsA across Caco-2 monolayers was assessed, a significant polarity in transport could be observed, the absorptive transport being much lower than the secretory transport. Inclusion of HIF resulted in a moderate increase of the absorptive transport, as well as a significant concentration dependent decrease of the secretory transport, without compromising the integrity of the monolayer. Interestingly, a possible gender difference could be detected as inclusion of HIF obtained from female subjects resulted in a decreased absorptive transport of CsA, whereas inclusion of HIF obtained from male subjects resulted in an increased absorptive transport. The P-gp modulating effect of HIF is not caused by a lack of glucose as an energy source for the efflux mechanism when high concentrations of HIF were present in the buffer used. Conclusions. The results of this study indicate that the contribution of P-gp efflux carriers may be overestimated when using salt buffer solutions as transport media. Additionally, it can be concluded that (presently unidentified) components of HIF may attenuate the P-gp mediated intestinal efflux. The clinical significance of this modulating effect remains to be investigated.     

Solid state properties of pure UC-781 and solid dispersions with polyvinylpyrrolidone (PVP K30)

The purpose of this study was to elucidate the physical structure of solid dispersions of the antiviral agent UC-781 (N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide) with polyvinylpyrrolidone (PVP K30). Solid dispersions were prepared by coevaporating UC-781 with PVP K30 from dichloromethane. The physicochemical properties of the dispersions were evaluated in comparison with the physical mixtures by differential scanning calorimetry (DSC), X-ray powder diffraction, and FT-IR spectroscopy. We investigated the single crystal structure of pure UC-781. The data from single crystal analysis showed that UC-781 crystallized with orthorhombic symmetry in the space group Pcab. Its cell parameters were found to be; a = 8.1556(7) A,b = 17.658(2) A and c = 23.609(2) A; the unit cell was made up of eight molecules of UC-781. The molecules formed intermolecular hydrogen bonds between NH and thio groups, and were packed in a herringbone-like structure. The data from X-ray powder diffraction showed that crystalline UC-781 was changed into the amorphous state by co-evaporating it with PVP K30. From differential scanning calorimetry analysis, UC-781 peaks were observed in the DSC curves of all physical mixtures, while no peaks corresponding to the drug could be observed in the solid dispersions with the same drug composition up to the concentration of 50% w/w. The data from FT-IR spectroscopy showed the distortions and disappearance of some bands from the drug, while other bands were too broad or significantly less intense compared with the physical mixtures of the crystalline drug in PVP K30. Furthermore, the results from IR spectroscopy demonstrated that UC-781 interacted with PVP K30 in solid dispersions through intermolecular H-bonding.     

Characterization of glassy itraconazole: a comparative study of its molecular mobility below T(g) with that of structural analogues using MTDSC

The objective of the present study was to estimate the molecular mobility of glassy itraconazole below the glass transition, in comparison with structural analogues (i.e. miconazole and ketoconazole).Glassy itraconazole and miconazole were prepared by cooling from the melt. The glassy state of the drug was investigated with modulated temperature DSC using the following conditions: amplitude +/-0.212 K, period 40 s, underlying heating rate 2 K/min. The glass transition was determined from the reversing heat flow and occurred at 332.4 (+/-0.5) K and 274.8 (+/-0.4) K for itraconazole and miconazole, respectively. The jump in heat capacity at the glass transition was 303.42 (+/-3.43) J/mol K for itraconazole and 179.35 (+/-0.89) J/mol K for miconazole. The influence of the experimental conditions on the position of the glass transition of itraconazole was investigated by varying the amplitude from +/-0.133 to +/-0.292 K and the period from 25 to 55 s, while the underlying heating rate was kept constant at 2 K/min. Glass transition temperature, T(g), was not significantly influenced by the frequency of the modulation nor by the cooling rate. However, the relaxation enthalpy at the glass transition increased with decreasing cooling rate indicating relaxation during the glass formation process. To estimate the molecular mobility of the glassy materials, annealing experiments were performed from T(g)--10 to T(g)--40 K for periods ranging from 15 min to 16 h.Fitting the extent of relaxation of glassy itraconazole to the Williams--Watts decay function and comparing the obtained values with those of amorphous miconazole and ketoconazole indicated that the molecular mobility is influenced by the complexity of the molecular structure. The more complex the structure, the more stable the amorphous state.     

Evaluation of the potential of ion pair formation to improve the oral absorption of two potent antiviral compounds, AMD3100 and PMPA.

9-(2-phosphonyomethoxypropyl)adenine (PMPA) and AMD3100 are highly potent and selective antiretroviral agents. Since PMPA is negatively charged and AMD3100 positively charged at physiological pH, their transepithelial transport and their potential for oral drug delivery is very low. In this study, ion pair formation was evaluated as a possible strategy to enhance transepithelial transport of PMPA and AMD3100. Positively charged counter ions such as t-hexyl-, t-heptyl-, t-octylammonium bromide and dodecyl-, tetradecyl-, hexadecyltrimethylammonium bromide were used to form ion pairs with PMPA, while sodium taurodeoxycholate (in vitro experiments) and sodium taurocholate (in vivo experiments) were used as counter ions for AMD3100. The effect of counter ions on transepithelial transport of PMPA (1 mM) and AMD3100 (1 mM) was investigated by measuring the flux across Caco-2 monolayers. An enhancement in drug transport could be observed at a concentration of 2 mM of hexadecyltrimethylammonium bromide (counter ion for PMPA) and 10 mM of sodium taurodeoxycholate (counter ion for AMD3100), but at the concentrations used, the absorption enhancing effect could be attributed to a reduction of the integrity of the monolayers. When AMD3100 transport was tested at a concentration of 200 microM, no flux was observed, even in the presence of relatively high concentrations of counter ion (20 times the concentration of AMD3100). Results obtained from partitioning studies of the drugs in the presence or absence of counter ion revealed that competition by other ions was responsible for the absence of an effect: when pure water was used as the aqueous phase, a reduction up to 24.4+/-1.4% and 17.0+/-1.3% of the initial aqueous concentration was observed for PMPA and AMD3100, respectively; however, as soon as other ions were present in the aqueous phase, the effect of the counter ion was diminished (25-50 mOsm) or completely abolished (270-305 mOsm). The absorption enhancing effect of counter ions was also studied in vivo: pharmacokinetic studies in rabbits showed that the oral bioavailability of AMD3100 in the presence of 4 equivalents of taurocholic acid remained very low and was only 3.2-fold better (i.e. 3.6%) in comparison to pure AMD3100. In view of the results obtained in the Caco-2 system, this absorption enhancement can be attributed to an effect on monolayer integrity rather than to the potential to form ion pairs. We can conclude that the formation of ion pairs may not be very efficient as a strategy to enhance transepithelial transport of charged hydrophilic compounds, as competition by other ions may abolish the beneficial effect of counter ions.