Victor Mangas-Sanjuan

Cyclometalated Iminophosphorane Gold(III) and Platinum(II) Complexes. A Highly Permeable Cationic Platinum(II) Compound with Promising Anticancer Properties.

New organometallic gold(III) and platinum(II) complexes containing iminophosphorane ligands are described. Most of them are more cytotoxic to a number of human cancer cell lines than cisplatin. Cationic Pt(II) derivatives 4 and 5, which differ only in the anion, Hg2Cl62– or PF6– respectively, display almost identical IC50 values in the sub-micromolar range (25–335-fold more active than cisplatin on these cell lines). The gold compounds induced mainly caspase-independent cell death, as previously reported for related cycloaurated compounds containing IM ligands. Cycloplatinated compounds 3, 4, and 5 can also activate alternative caspase-independent mechanisms of death. However, at short incubation times cell death seems to be mainly caspase dependent, suggesting that the main mechanism of cell death for these compounds is apoptosis. Mercury-free compound 5 does not interact with plasmid (pBR322) DNA or with calf thymus DNA. Permeability studies of 5 by two different assays, in vitro Caco-2 monolayers and a rat perfusion model, have revealed a high permeability profile for this compound (comparable to that of metoprolol or caffeine) and an estimated oral fraction absorbed of 100%, which potentially makes it a good candidate for oral administration.

Ion-pair strategy for enabling amifostine oral absorption: rat in situ and in vivo experiments.

This study shows the effect of ion pair formation on intestinal absorption and oral bioavailability of amifostine. Amifostine is a prodrug used as a highly potent and selective radiotherapy and chemotherapy protectant but due to its low lipophilicity and charge at physiological pH range, its trans epithelial transport and its potential for oral drug delivery is very low. Ion pair formation with negatively charged counter ions was evaluated by in situ rat perfusion studies as a possible strategy to enhance intestinal absorption of amifostine. Succinic acid, phthalic acid and benzoic acid were used as counter ions. Rat intestinal perfusion studies confirmed a statistically significant increase in amifostine permeability in the presence of the counter ions in the order of succinic>phthalic>benzoic. Rat pharmacokinetic studies in vivo were performed to calculate oral absolute bioavailability of amifostine alone and with ion pairs in order to confirm the in situ perfusion results and the applicability of the ion pair approach. Intravenous and intraduodenal administrations were done in rats using a permanent jugular vein cannulation technique and a duodenal cannulation method to avoid drug degradation in stomach. In vivo oral bioavailability studies demonstrated a 20-30-fold increase in amifostine bioavailability with succinic acid depending on counter ion ratio and 10-fold increase with phthalic acid as ion pair. In summary ion pair strategy with succinic acid could enable amifostine oral administration on enteric coated formulations.

Tubulin acetylation promoting potency and absorption efficacy of deacetylase inhibitors.

Histone deacetylase 6 (HDAC6) and silent information regulator 2 (SIRT2) control the dynamics of the microtubule network via their deacetylase activities. Tubulin polymerization promoting protein (TPPP/p25) enhances microtubule acetylation by its direct binding to HDAC6. Our objective was to characterize the multiple interactions of the deacetylases and to establish the inhibitory potency and the pharmacokinetic features of the deacetylase inhibitors, trichostatin A (TSA) and AGK2.

Innovative in vitro method to predict rate and extent of drug delivery to the brain across the blood-brain barrier.

The relevant parameters for predicting rate and extent of access across the blood-brain barrier (BBB) are fu,plasma (unbound fraction in plasma), Vu,brain (distribution volume in brain) and Kp,uu,brain (ratio of free concentrations in plasma and brain). Their estimation still requires animal studies and in vitro low throughput experiments which make difficult the screening of new CNS candidates. The aim of the present work was to develop a new whole in vitro high throughput method to predict drug rate and extent of access across the BBB. The system permits estimation of fu,plasma, Vu,brain and Kp,uu,brain in a single experimental system, using in vitro cell monolayers in different conditions. From the ratios of the apparent permeability values (Papp) with the adequate mathematical analysis the relevant parameters can be estimated. Papp of ten model compounds has been obtained in MDCKII and MDCK-Mdr1cell monolayers in the absence and presence of albumin and brain homogenate. The ratio of Papp in the absence and presence of albumin allows estimation of in vitro fu,plasma. Papp in the presence of brain homogenate is used to estimate fu,brain and Vu,brain. Kp,uu,brain is estimated from the apical to basal versus basal to apical clearances. The BBB parameters obtained with the new method were predictive of the in vivo behavior of candidates. In vitro fu,plasma, Kp,uu,brain and Vu,brain (calculated with Papp from MDCKII cell line) presented a good correlation with in vivo fu,plasma, Kp,uu,CSF and Vu,brain published values (r=0.92; r=0.85; and r=0.99 respectively). Despite its simplicity the predictive performance is fairly good considering the reduced number of tested compounds with different physicochemical and transport properties. Further experimental modifications could be checked to optimize the method, but the present data support its feasibility. As other in vitro cell culture models, the system is suitable for miniaturization and robotization to allow high throughput screening of CNS candidates.

Semisynthesis, Cytotoxic Activity, and Oral Availability of New Lipophilic 9-Substituted Camptothecin Derivatives

Despite that 9-substituted camptothecins are promising candidates in cancer therapy, the limited accessibility to this position has reduced the studies of these derivatives to a few standard modifications. We report herein a novel semisynthetic route based on the Tscherniac-Einhorn reaction to synthesize new lipophilic camptothecin derivatives with amidomethyl and imidomethyl substitutions in position 9. Compounds were evaluated for their antiproliferative activity, topoisomerase I inhibition, and oral availability. Preliminary data demonstrated that bulky imidomethyl modification is an appropriate lipophilic substitution for an effective oral administration relative to topotecan. In addition, this general procedure paves the way for obtaining new camptothecin derivatives.

Variability of permeability estimation from different protocols of subculture and transport experiments in cell monolayers

INTRODUCTION In vitro models with high predictive ability have been revealed as strong tools for pharmaceutical industry. However, the variability in permeability estimations complicates the comparison and combination of data from different laboratories and it makes necessary the careful validation of the model and the continuous suitability demonstration. The adequate standardization of pre-experimental, experimental and post-experimental factors might help to reduce the inter- and intra-laboratory variability in permeability values. METHODS The objective of this paper is the evaluation of the effect of passage number, experimental protocol, time after seeding and calculation method on the permeability values and their variability in transport experiments in Caco-2, MDCK and MDCK-MDR1 cells. Metoprolol, Lucifer yellow and Rhodamine-123 were used to check the performance of the cell lines. Protocols used differ mainly in the differentiation time and the filter support coating with collagen. Data was analyzed with sink and non-sink approaches. The final purpose was to explore pre-experimental, experimental and post-experimental conditions in order to select the best experimental scenarios for permeability assays. RESULTS Results indicated that for passive diffusion studies, coating helps cell differentiation in a more stable manner in all cell lines compared to protocol without coating which showed permeability changes with passages and more variable values. In both protocols the paracellular route became more restricted with higher passage numbers. Functionality of P-gp assessed with Rhodamine permeability did not change with passage number in Caco-2 cells with any of the protocols but increased in both protocols in MDCK and MDCK-MDR1 cells. Protocol without coating showed the less variable results in these cell lines. Rhodamine permeabilities increased with higher maturation times due to a higher expression of the transporter. Nevertheless for compounds absorbed by passive diffusion there was not a clear trend neither in permeability values nor in variability. DISCUSSION As a conclusion, we have confirmed the influence of maturation conditions and passage number in permeability values and in their variability. Based on our results protocol with coating would be more adequate for studies of compounds absorbed by passive diffusion but the protocol without coating gave us better results for studies about P-gp interactions.

In vitro–in vivo correlations: general concepts, methodologies and regulatory applications

Abstract The major objective of in vitro–in vivo correlations is to be able to use in vitro data to predict in vivo performance serving as a surrogate for an in vivo bioavailability test and to support biowaivers. Therefore, the aims of this review are: (i) to clarify the factors involved during bio-predictive dissolution method development; and (ii) the elements that may affect the mathematical analysis in order to exploit all information available. This article covers the basic aspects of dissolution media and apparatus used in the development of in vivo predictive dissolution methods, including the latest proposals in this field as well as the summary of the mathematical methods for establishing the in vitro–in vivo relationship and their scope and limitations. The incorporation of physiological relevant factors in the in vitro dissolution method is essential to get accurate in vivo predictions. Standard quality control dissolution methods do not necessarily reflect the in vivo behavior, so they rarely are useful for predicting in vivo performance. The combination of physiological based dissolution methods with physiological-based pharmacokinetics models incorporating gastrointestinal variables will lead to robust tools for drug and formulation development, nevertheless their regulatory use for biowaiver application still require harmonization of the mathematical methods proposed and more detailed recommendations about the procedures for setting up dissolution specifications.

Assessment of the Regulatory Methods for the Comparison of Highly Variable Dissolution Profiles.

The objective is to compare the performance of dissolution-profile comparison methods when f2 is inadequate due to high variability. The 90% confidence region of the Mahalanobis distance and the 90% bootstrap confidence interval (CI) of the f2 similarity factor (f2-bootstrap) were explored. A modification of the Mahalanobis distance (new D-Mahalanobis) in which those points >85% were not taken into account for calculation was also used. A population kinetic approach in NONMEM was used to simulate dissolution profiles with the first-order or Weibull kinetic models. The scenarios were designed to have clearly similar, clearly non-similar or borderline situations. Four different conditions of variability were established: high (CV = 20%) and low variability (CV = 5%) for inter-tablet (IIV) and inter-batch variability (IBV) associated to the dissolution parameters (kd or MDT) using an exponential model. Forty-four (44) scenarios were simulated, considering different combinations of IIV, IBV and typical dissolution parameters. The dissolution profiles simulated using a first-order model modified the profile slope. The Weibull model allows profiles with different shapes and asymptotes and crossing each other. The results show that the f2-bootstrap is the most adequate method in cases of high variability. The method based on the 90% confidence region of the Mahalanobis distance (D-Mahalanobis) is not able to detect large differences that can be detected simply with f2 (i.e. low specificity and positive predictive value due to false positives). The new D-Mahalanobis exhibits superior sensitivity to detect differences (i.e. specificity as a diagnostic test), but it is not as good as the f2-bootstrap method.

Semimechanistic Cell-Cycle Type–Based Pharmacokinetic/Pharmacodynamic Model of Chemotherapy-Induced Neutropenic Effects of Diflomotecan under Different Dosing Schedules

The current work integrates cell-cycle dynamics occurring in the bone marrow compartment as a key element in the structure of a semimechanistic pharmacokinetic/pharmacodynamic model for neutropenic effects, aiming to describe, with the same set of system- and drug-related parameters, longitudinal data of neutropenia gathered after the administration of the anticancer drug diflomotecan (9,10-difluoro-homocamptothecin) under different dosing schedules to patients (n = 111) with advanced solid tumors. To achieve such an objective, the general framework of the neutropenia models was expanded, including one additional physiologic process resembling cell cycle dynamics. The main assumptions of the proposed model are as follows: within the stem cell compartment, proliferative and quiescent cells coexist, and only cells in the proliferative condition are sensitive to drug effects and capable of following the maturation chain. Cell cycle dynamics were characterized by two new parameters, FProl (the fraction of proliferative [Prol] cells that enters into the maturation chain) and kcycle (first-order rate constant governing cell cycle dynamics within the stem cell compartment). Both model parameters were identifiable as indicated by the results from a bootstrap analysis, and their estimates were supported by date from the literature. The estimates of FProl and kcycle were 0.58 and 1.94 day−1, respectively. The new model could properly describe the neutropenic effects of diflomotecan after very different dosing scenarios, and can be used to explore the potential impact of dosing schedule dependencies on neutropenia prediction.

Drug gastrointestinal absorption in rat: Strain and gender differences

Predictive animal models of intestinal drug absorption are essential tools in drug development to identify compounds with promising biopharmaceutical properties. In situ perfusion absorption studies are routinely used in the preclinical setting to screen drug candidates. The objective of this work is to explore the differences in magnitude and variability on intestinal absorption associated with rat strain and gender. Metoprolol and Verapamil absorption rate coefficients were determined using the in situ closed loop perfusion model in four strains of rats and in both genders. Strains used were Sprague-Dawley, Wistar-Han, Wistar-Unilever, Long-Evans and CD∗IGS. In the case of Metoprolol only CD∗IGS and Wistar Unilever showed differences between males and females. For Verapamil, Wistar Han and Sprague-Dawley strains do not show differences between male and female rats. That means that in these strains permeability data from male and female could be combined. In male rats, which are commonly used for permeability estimation, there were differences for Metoprolol permeability between Sprague-Dawley (with lower permeability values) and the other strains, while for Verapamil Sprague-Dawley and Wistar-Han showed the lower permeability values. In conclusion, the selection of rats strain and gender for intestinal absorption experiments is a relevant element during study design and data from different strains may not be always comparable.