Stereospecific chemical and enzymatic stability of phosphoramidate triester prodrugs of d4T in vitro.

The phosphoramidate triester prodrug approach is widely used to deliver nucleotide forms of nucleoside analogues into target cells. We investigated the stereoselective stability of a series of prodrugs of the anti-HIV agent 2',3'-didehydro-2',3'-dideoxythymidine (d4T). Chemical stability was evaluated in phosphate buffer at pH values of biological relevance (i.e. pH 2.0, 4.6, 7.4). Enzymatic stability was tested in human plasma, in Caco-2 cell homogenates and monolayers and in rat liver. The compounds were relatively stable to chemical hydrolysis. Between 50 and 70% of unchanged prodrug was recovered after 16h incubation in human plasma, with no stereoselective preference for phosphate diastereoisomers. The p-OMe phenyl derivative, however, was an exception and only 5% of one diastereoisomer was recovered. In Caco-2 cells the stability and stereoselectivity largely depended on the experimental conditions: high enzymatic activity and stereoselectivity was observed in cell homogenates, but not in monolayers. In rat liver S9 fractions the stability profile was similar to that in Caco-2 cells and carboxyl ester cleavage appeared to be the sole mechanism of degradation in both media. The large and unpredictable differences in stereoselective metabolic rate of the pronucleotide series here presented suggest that in vivo circulating levels of intact prodrug could exert profoundly different activity or toxicity due to preferential body distribution of one diastereoisomeric form.     

Endocytic Uptake,Transport and Macromolecular Interactions of Anionic PAMAM Dendrimers within Lung Tissue

Purpose

Polyamidoamine (PAMAM) dendrimers are a promising class of nanocarrier with applications in both small and large molecule drug delivery. Here we report a comprehensive evaluation of the uptake and transport pathways that contribute to the lung disposition of dendrimers.

Methods

Anionic PAMAM dendrimers and control dextran probes were applied to an isolated perfused rat lung (IPRL) model and lung epithelial monolayers. Endocytosis pathways were examined in primary alveolar epithelial cultures by confocal microscopy. Molecular interactions of dendrimers with protein and lipid lung fluid components were studied using small angle neutron scattering (SANS).

Results

Dendrimers were absorbed across the intact lung via a passive, size-dependent transport pathway at rates slower than dextrans of similar molecular sizes. SANS investigations of concentration-dependent PAMAM transport in the IPRL confirmed no aggregation of PAMAMs with either albumin or dipalmitoylphosphatidylcholine lung lining fluid components. Distinct endocytic compartments were identified within primary alveolar epithelial cells and their functionality in the rapid uptake of fluorescent dendrimers and model macromolecular probes was confirmed by co-localisation studies.

Conclusions

PAMAM dendrimers display favourable lung biocompatibility but modest lung to blood absorption kinetics. These data support the investigation of dendrimer-based carriers for controlled-release drug delivery to the deep lung.

     

Enhanced pulmonary absorption of a macromolecule through coupling to a sequence-specific phage display-derived peptide

With the aim of identifying a peptide sequence that promotes pulmonary epithelial transport of macromolecule cargo we used a stringent peptide-phage display library screening protocol against rat lung alveolar epithelial primary cell cultures. We identified a peptide-phage clone (LTP-1) displaying the disulphide-constrained 7-mer peptide sequence, C-TSGTHPR-C, that showed significant pulmonary epithelial translocation across highly restrictive polarised cell monolayers. Cell biological data supported a differential alveolar epithelial cell interaction of the LTP-1 peptide-phage clone and the corresponding free synthetic LTP-1 peptide. Delivering select phage-clones to the intact pulmonary barrier of an isolated perfused rat lung (IPRL) resulted in 8.7% of lung deposited LTP-1 peptide-phage clone transported from the IPRL airways to the vasculature compared (p < 0.05) to the cumulative transport of less than 0.004% for control phage-clone groups. To characterise phage-independent activity of LTP-1 peptide, the LTP-1 peptide was conjugated to a 53 kDa anionic PAMAM dendrimer. Compared to respective peptide-dendrimer control conjugates, the LTP-1-PAMAM conjugate displayed a two-fold (bioavailability up to 31%) greater extent of absorption in the IPRL. The LTP-1 peptide-mediated enhancement of transport, when LTP-1 was either attached to the phage clone or conjugated to dendrimer, was sequence-dependent and could be competitively inhibited by co-instillation of excess synthetic free LTP-1 peptide. The specific nature of the target receptor or mechanism involved in LTP-1 lung transport remains unclear although the enhanced transport is enabled through a mechanism that is non-disruptive with respect to the pulmonary transport of hydrophilic permeability probes. This study shows proof-of principle that array technologies can be effectively exploited to identify peptides mediating enhanced transmucosal delivery of macromolecule therapeutics across an intact organ.     

Percutaneous Penetration Kinetics of Nitroglycerin and Its Dinitrate Metabolites Across Hairless Mouse Skin in Vitro

The percutaneous penetration kinetics of the antianginal, nitroglycerin (GTN), and its primary metabolites, 1,2- and 1,3-glyceryl dinitrate (1,2- and 1,3-GDN), were evaluated in vitro, using full-thickness hairless mouse skin. GTN and the 1,2- and 1,3-GDNs were applied (a) in aqueous solution as pH 7.4 phosphate-buffered saline (PBS) and (b) incorporated into lipophilic ointment formulations. The cutaneous transformation of GTN to its dinitrate metabolites was detected, but no interconversion between 1,2-GDN and 1,3-GDN was observed. Following application of the nitrates in PBS solution, all three compounds exhibited steady-state transport kinetics. The steady-state flux of GTN (8.9 ± 1.5 nmol cm^–2 hr^–1) was significantly greater (P < 0.05) than those of 1,2-GDN (0.81 ± 0.54 nmol cm^–2 hr^–1) and 1,3-GDN (0.72 ± 0.20 nmol cm^–2 hr^–1). The corresponding permeability coefficient () for GTN (20 ± 3 × 10^–3 cm hr^–1) was significantly larger than the corresponding values for 1,2-GDN (1.4 ± 0.9 × 10^–3 cm hr^–1) and 1,3-GDN (1.2 ± 0.4 × 10^–3 cm hr^–1), which were statistically indistinguishable (P > 0.05). Further analysis of the transport data showed that the differences between GTN and the GDNs could be explained by the relative stratum corneum/water partition coefficient (K _s) values of the compounds. The apparent partition parameters, defined as = K _s · h [where h is the diffusion path length through stratum corneum (SC)] were 19.8 ± 2.5 × 10^–2 cm for GTN and 1.91 ± 1.07 × 10^–2 and 1.81 ± 0.91 × 10^–2 cm for 1,2- and 1,3-GDN, respectively. However, when the nitrates were administered in an ointment base, the apparent partition parameter (') and permeability coefficient (') of GTN markedly decreased, to 2.51 ± 0.75 × 10^–2 cm and 1.6 ± 0.3 × 10^–3 cm hr^–1, respectively. In contrast, the ' and ' results for 1,2- and 1,3-GDN were not significantly different (P > 0.05) from the corresponding and values, which were measured following dosing as aqueous solutions. As a result, the steady-state fluxes of all three nitrates from the ointment formulation were comparable (GTN, 154 ± 28 nmol cm^–2 hr^–1; 1,2-GDN, 162 ± 22 nmol cm^–2 hr^–1; 1,3-GDN, 162 ± 34 nmol cm^–2 hr^–1). It follows that the dinitrates can be as efficiently delivered across the skin as GTN when a suitable formulation is employed. This finding may support transdermal therapy using 1,2- or 1,3-GDN if, indeed, they are found to be pharmacologically effective.     

Caveolin expression during chondrogenesis in the avian limb.

Caveolin is the principal component and critical structural and functional element of caveolae, omega-shaped plasmalemmal invaginations, which have been implicated in a wide range of cellular processes in several different tissues. In the present study, we have investigated both the spatial and temporal expression of caveolin proteins during chondrogenesis in the avian tibiotarsus at days 10-20 of embryonic development. By using semiquantitative Western blotting, we found that caveolin-1 was clearly expressed in developing avian cartilage. The positive expression of caveolin-1 in cartilage showed an upward trend of accumulation temporally, with the highest levels of expression at 20 days of development. By using immunocytochemistry, we detected all three caveolin proteins in the cells of the outer fibrous articular surface, although caveolin-1 demonstrated the strongest and most consistent reactivity. In all cases, however, immunoreactivity appeared to be concentrated in cells facing the articular cavity. In the epiphyseal cartilage, immunocytochemistry revealed that caveolin-1 was present in the majority of chondrocytes within all layers of the cartilage and at all stages examined. A discrete, intense band of caveolin-1 immunoreactivity was apparent within the layer of flattened cells immediately underlying the proliferating rounded chondrocytes and suggests that caveolin-1 might be involved in regulating the progression of cells through these gradually maturing cell layers. In contrast to the results for caveolin-1, in the case of caveolin-2 and -3, chondrocytes were devoid of immunoreactivity in all regions of the epiphyseal cartilage. Overall, this study demonstrates that caveolin-1, -2, and -3 are expressed during chondrogenesis in the developing avian limb, although the patterns of expression are restricted both spatially and temporally throughout the differentiating cell layers of the cartilage. The results suggest that caveolin proteins might play a differentiation-dependent role during avian chondrogenesis.     

The Differential Absorption of a Series of P-Glycoprotein Substrates in Isolated Perfused Lungs from Mdr1a/1b Genetic Knockout Mice can be Attributed to Distinct Physico-Chemical Properties: an Insight into Predicting Transporter-Mediated,Pulmonary Specific Disposition

Purpose

To examine if pulmonary P-glycoprotein (P-gp) is functional in an intact lung; impeding the pulmonary absorption and increasing lung retention of P-gp substrates administered into the airways. Using calculated physico-chemical properties alone build a predictive Quantitative Structure-Activity Relationship (QSAR) model distinguishing whether a substrate’s pulmonary absorption would be limited by P-gp or not.

Methods

A panel of 18 P-gp substrates were administered into the airways of an isolated perfused mouse lung (IPML) model derived from Mdr1a/Mdr1b knockout mice. Parallel intestinal absorption studies were performed. Substrate physico-chemical profiling was undertaken. Using multivariate analysis a QSAR model was established.

Results

A subset of P-gp substrates (10/18) displayed pulmonary kinetics influenced by lung P-gp. These substrates possessed distinct physico-chemical properties to those P-gp substrates unaffected by P-gp (8/18). Differential outcomes were not related to different intrinsic P-gp transporter kinetics. In the lung, in contrast to intestine, a higher degree of non-polar character is required of a P-gp substrate before the net effects of efflux become evident. The QSAR predictive model was applied to 129 substrates including eight marketed inhaled drugs, all these inhaled drugs were predicted to display P-gp dependent pulmonary disposition.

Conclusions

Lung P-gp can affect the pulmonary kinetics of a subset of P-gp substrates. Physico-chemical relationships determining the significance of P-gp to absorption in the lung are different to those operative in the intestine. Our QSAR framework may assist profiling of inhaled drug discovery candidates that are also P-gp substrates. The potential for P-gp mediated pulmonary disposition exists in the clinic.

     

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Caveolin-1 displays both tumour-suppressor and tumour-promoter properties in breast cancer. Using characterised preclinical cell models for the transition of oestrogen-sensitive (WT-MCF-7 cells) to a tamoxifen-resistant (TAM-R cells) phenotype we examined the role caveolin-1 in the development of hormone-resistant breast cancer. The WT-MCF-7 cells showed abundant expression of caveolin-1 which potentiated oestrogen-receptor (ERα) signalling and promoted cell growth despite caveolin-1 mediating inhibition of ERK signalling. In TAM-R cells caveolin-1 expression was negligible, repressed by EGF-R/ERK signalling. Pharmacological inhibition of EGFR/ERK in TAM-R cells restored caveolin-1 and also resulted in the emergence of pools of phosphorylated caveolin-1. WT-MCF-7 cells exposed to tamoxifen for upto 12 weeks displayed increased caveolin-1 (peaking by week 2) followed (after week 8) by a marked decrease as the cells progress to develop a stable tamoxifen-resistant phenotype. The targeted down-regulation (siRNA) of caveolin-1 in WT-MCF-7 cells reduced growth but did not affect their sensitivity to tamoxifen, suggesting loss of caveolin-1 alone is not sufficient to confer tamoxifen-resistance. Hyperactivation of EGFR/ERK is a feature of tamoxifen-resistant breast cancer cells, a principal driver of cell growth. Recombinant expression of caveolin-1 in TAM-R cells did not affect EGFR/ERK activity, potentially due to mislocalisation of caveolin-1 through hyperactivation of the mTOR pathway or altered caveolin-1 phosphorylation. This work defines a novel role for caveolin-1 with implications for the clinical course of breast cancer and identifies caveolin-1 as a potential drug target for the treatment of early oestrogen-dependent breast cancers. Further, the loss of caveolin-1 may have benefit as a molecular signature for tamoxifen resistance.