Determination of intraliposomal pH and its effect on membrane partitioning and passive loading of a hydrophobic camptothecin, DB-67

The purpose of this work was to study the effect of pH on the liposomal encapsulation of a model camptothecin anti-tumor agent, DB-67, by considering the state of ionization and bilayer membrane/water partitioning of the drug as a function of pH. A novel fluorescence method was developed to monitor intravesicular pH in liposomal formulations containing entrapped DB-67 by using the drug itself as a pH indicator. Fluorescence spectra were recorded in aqueous buffers and liposomes and used to estimate the ionization constant of the A-ring phenol of DB-67 (pKappa(a2)) and shifts in ionization constants ( pKappa (a1) and pKappa(a2) ) due to membrane binding. Bilayer/water partitioning studies by equilibrium dialysis were employed to show that DB-67 is highly membrane bound over the entire pH range examined though binding decreases with an increase in pH. The observed ionization constants of membrane-bound DB-67 obtained from the equilibrium dialysis experiments were consistent with observations from fluorescence measurements and previous permeability results. The pH dependence of DB-67 loading using a passive loading technique was found to reflect the pH dependence of membrane binding of the drug. This results in poor encapsulation efficiency of DB-67 at high pH, necessitating further development of formulation strategies to improve loading efficiency.     

Effect of quinidine on membrane properties: Depression of the lipid phase transition temperature and changes in the permeability of the lipid bilayer

The influence of an antiarrhythmic drug, quinidine, on the physical state of membrane phospholipids was investigated using model membranes, liposomes. Turbidimetric measurements on liposomes prepared from neutral (dipalmitoyl phosphatidylcholine) and acidic (dipalmitoyl phosphatidic acid) phospholipids showed that quinidine reduces the temp of the gel to liquid-crystalline phase transition and broadens the temp range of the transition. The effect of quinidine on the thermal behaviour of model membranes depends on both the pH and the type of phospholipids used. It is markedly stronger for acidic than for neutral phospholipids, suggesting the importance of electrostatic effects in drug-membrane interaction. The ability of quinidine to interact with the lipid bilayer was confirmed by permeability measurements with the use of a self-quenched fluorescent compound, calcein. It is suggested that quinidine-phospholipid interaction may contribute to the mechanisms by which the drug exerts its physiological and pharmacological effects.     

Sustained release of hydrophobic and hydrophilic drugs from a floating dosage form

Floating dosage forms enable the sustained delivery of drugs in the gastro-intestinal tract. In this study, a type of multi-unit floating gel bead was synthesized with calcium alginate, sunflower oil, and a drug of interest through an emulsification/gelation process. The alginate beads with oil addition were able to continuously float over the medium for 24h under constant agitation while the non-oily beads could not. Three kinds of drugs with different hydrophilicities, ibuprofen, niacinamide and metoclopramide HCl, were tested in the study. The hydrophobic drug ibuprofen was released in a sustained manner for 24h, due to the oil partitioning. With suitable modification, the beads were able to also release the hydrophilic drugs, niacinamide and metoclopramide HCl, for a similar duration. Therefore a floating dosage form that is able to sustain release both hydrophobic and hydrophilic drugs within its extended gastric retention time has been developed.     

Novel stable camptothecin derivatives replacing the E-ring lactone by a ketone function are potent inhibitors of topoisomerase I and promising antitumor drugs

The E-ring lactone is the Achilles' heel of camptothecin derivatives: although it is considered necessary for the inhibition of the enzyme topoisomerase I (topo1), the opening of the lactone into a carboxylate abolishes the generation of topo1-mediated DNA breaks. S38809 is a novel camptothecin analog with a stable 5-membered E-ring ketone; therefore, it lacks the lactone function. DNA relaxation and cleavage assays revealed that S38809 functions as a typical topo1 poison by stimulating DNA cleavage at T downward arrow G sites. The activity was strongly dependent on the stereochemistry of the C-7 carbon atom that bears the hydroxy group. S38809 proved to be a potent cytotoxic agent, with a mean IC50 of 5.4 nM versus 11.6 nM for topotecan and 3.3 nM for SN38 (the active metabolite of irinotecan) on a panel of 31 human tumor cell lines. The cytotoxicity of S38809 and its ability to stabilize cleavable complexes was considerably reduced in camptothecin-resistant cells that express a mutated topo1, confirming that topo1 is its primary target. Cell death induced by topo1 poisoning requires the conversion of DNA single-strand breaks into double-strand breaks that can be detected by the formation of phosphorylated histone H2AX. In HCT116 cells, topotecan, SN38, and S38809 induced histone H2AX phosphorylation in S phase of the cell cycle, with S38809 being as potent as SN38 and 5-fold more potent than topotecan. In vivo, S38809 showed a marked antitumor activity against HCT116 xenografts. These findings open a new route for improving the pharmacological properties of camptothecin derivatives.     

Caco-2 permeability,P-glycoprotein transport ratios and brain penetration of heterocyclic drugs

In this study the gastrointestinal absorption and P-glycoprotein (Pgp) efflux transport of heterocyclic drugs was investigated with the Caco-2 cell model. Based on the calculation of the physico-chemical properties a good oral absorption was predicted for all the drugs tested in this study which corresponded well with the measured Caco-2 permeabilities (Papp). Generally a high permeability of the tested heterocyclic drugs was measured being in agreement with earlier published human in vivo absorption data. Based on the transport data of domperidone and verapamil it was found that the Pgp efflux transporter was expressed in the Caco-2 cells. Many of the drugs tested were indicated to be potential Pgp efflux substrates. Since Pgp is expressed at the Blood Brain Barrier (BBB) as well, it was expected that CNS penetration will be impaired if a drug is a Pgp substrate. However, no correlation could be found between brain penetration in rats and the Pgp efflux ratio as measured with the Caco-2 cells. From the data it is concluded that Pgp efflux ratio's as determined in in vitro High Throughput Screening (HTS) tests, where the transport conditions are fixed (pH gradient, concentration, etc.), cannot routinely be used to predict a possible limited brain penetration.     

New lipid modulating drugs: the role of microsomal transport protein inhibitors

Microsomal triglyceride transfer protein (MTP) is involved in the synthesis of very low density lipoprotein in the liver. Its deficiency results in abetalipoproteinemia. MTP inhibitors target the assembly and secretion of apolipoprotein B-containing lipoproteins. These agents may potentially play a role, alone or in combination, in the treatment of hypercholesterolemia or hypertriglyceridaemia. Clinical applications of MTP inhibitors initially focused primarily on high-dose monotherapy in order to produce substantial reductions in LDL-cholesterol levels but these proved to induce significant hepatic steatosis and transaminase elevations. However, likely orphan indications for MTP inhibitors, where a different risk-benefit profile applies, include patients with homozygous familial hypercholesterolemia where statins often show a low response. Development of MTP inhibitors has continued to enter clinical trials at lower doses or in formulations aimed at utilizing their efficacy while avoiding their side effects. These have shown promising results in reducing cholesterol, triglycerides and apolipoprotein B with a far lower incidence of, often, transient side-effects. The clinical efficacy and safety of MTP inhibition in patients with hyperlipidaemia remains to be fully determined and to be proven in both surrogate and clinical endpoint trials but there may be a role for these agents in orphan indications for rarer severe hyperlipidaemias.     

Solvation of drugs as a key for understanding partitioning and passive transport exemplified by NSAIDs

Passive transport properties of drug molecules are of utmost importance for their pharmacological and biopharmaceutical effectiveness. Diffusion in different media and through lipid bilayers is in many cases the rate-determining step for the distribution in the body. In the present review an attempt is made to demonstrate the importance of solvation of drug molecules for the diffusion and partition/distribution in phases of different lipophilicity. Different approaches known in the literature to describe solvation of compounds with flexible conformation are discussed as well as the experimental methods to directly measure the energy of solvation. NSAIDs are chosen as an example of a class of drugs of different molecular structures that have already been studied thoroughly in many aspects, and a set of aliphatic alcohols can be used as a model for compartments of different lipophilic/hydrophilic properties. Thermodynamic characteristics of solvation of the drug molecules yielded by independent classical experimental methods (Gibbs energy, enthalpic and entropic terms of Gibbs energy) are studied in order to better understand diffusion and distribution properties. Correlations between in-vitro-data (partition coefficient, enthalpy of solvation) with biopharmaceutically relevant characteristics (plasma half-life) are also discussed.     

Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity

Several camptothecin derivatives containing a modified hydroxy lactone ring have been synthesized and evaluated for inhibition of topoisomerase I and cytotoxicity to mammalian cells. Each of the groups of the hydroxy lactone moiety, the carbonyl oxygen, the ring lactone oxygen, and the 20-hydroxy group, were shown to be critical for enzyme inhibition. For example the lactol, lactam, thiolactone, and 20-deoxy derivatives did not stabilize the covalent DNA-topoisomerase I complex. With a few exceptions, those compounds that did not inhibit topoisomerase I were not cytotoxic to mammalian cells. Two cytotoxic derivatives that did not inhibit topoisomerase I were shown to produce non-protein-associated DNA single-strand breaks and are likely to have a different mechanism of action. One of these compounds was tested for antitumor activity and was found to be inactive. The present findings, as well as other reports that the hydroxy lactone ring of camptothecin is critical for antitumor activity in vivo, correlate with the structure-activity relationships at the level of topoisomerase I and support the hypothesis that antitumor activity is related to inhibition of this target enzyme.     

Feasibility studies of dermal delivery of paclitaxel with binary combinations of ethanol and isopropyl myristate: role of solubility, partitioning and lipid bilayer perturbation

In the current investigation, paclitaxel (PCL) delivery into the different layers of skin, vehicle optimization and relationship between vehicle composition and the relative contribution of solubility, partition and diffusion towards drug transport has been outlined. Saturation solubility of PCL was determined in ethanol (EtOH), isopropyl myristate (IPM) and their binary combinations, and partition studies performed to study the probability of skin depot formation. Epidermal and dermal partitioning was carried from PCL saturated vehicles. Skin permeation of PCL was studied using the rat skin. FT-IR has been utilized to study the skin barrier perturbation, and the localization of PCL and isopropyl myristate (IPM) in epidermis. High K(app) value in mineral oil/buffer indicated the tendency of PCL to form a reservoir in skin, and an inverse relationship between PCL solubility in different solvent systems and partitioning into epidermis was found. Maximum K(epidermis) for PCL was observed with IPM, while PCL in EtOH/IPM (1:1) showed high partitioning into dermis. Maximum flux of PCL was observed with EtOH/IPM (1:1). For lipophilic drug like PCL modulation of vehicle seems to be effective approach to increase the permeability across the skin. With a binary combination of EtOH/IPM (1:1) higher concentration of PCL can be delivered to deeper layer of skin whereas with IPM higher concentration of PCL could be localized in the epidermis. While engineering the delivery vehicle selection of solvents should be such that one of them is miscible in both hydrophilic and lipophilic phase like ethanol and another should be lipophilic in nature (IPM in this case) so that an optimum balance between 'push-pull' and 'blending' effect can be achieved.     

Determination of total and lactone form of a new camptothecin derivative gimatecan (ST1481) and its metabolite ST1698 in human plasma by high-performance liquid chromatography with fluorimetric detection

A new sensitive high-performance liquid chromatographic (HPLC) method for the determination of gimatecan (ST1481), a new camptothecin derivative, and its metabolite (ST1698) in plasma sample has been developed. The method consisted of on-line column solid phase extraction of analytes from human plasma, chromatographic separation by isocratic elution, then fluorimetric detection. The limits of quantitation were 0.25 ng/mL for both the analytes. The recovery of the extraction procedure was in the range of 62.8–71.1% for all the compounds. Good linearity (R² > 0.999) was observed within the calibration ranges studied: 0.25–25 ng/mL for both ST1481 and ST1698. Precision was in the range 1.2–4.3%, and accuracy was always lower than 4.7%. Surprisingly, after administration of ST1481 to humans, plasma concentrations found were higher than expected, while metabolite plasma concentrations were negligible. For this reason, a second calibration curve range was validated to quantify ST1481 in human plasma, ranging from 5 to 200 ng/mL. A good accuracy and precision were obtained, confirming the usefulness of the procedure. By using neutral analytical condition the intact lactone form was estimated in plasma samples from a patient. The lactone form amounted to 80–100% of the total ST1481.     

Rapid communication: partitioning of persistent lipophilic compounds,including dioxins,between human milk lipid and blood lipid: an initial assessment

A systematic program of sampling and analysis of blood serum for dioxins, furans, and dioxinlike polychlorinated biphenyls (PCBs) has been initiated in the United States through the National Health and Nutrition Examination Survey (NHANES) program. While such data could potentially be used to estimate population-level changes in human milk lipid concentrations of chemicals, such estimates would depend on understanding the relationship between human blood lipid and milk lipid concentrations of the compounds of interest. For dioxins and furans, extremely limited data in humans currently exist for paired blood/milk samples. These data reviewed in this article, support the hypothesis that, over a population and across time, human milk lipid levels of these compounds generally reflect blood lipid levels. However, these data also suggest that significant variations in these ratios are possible among individuals and at various times.     

The dependence of the lipid bilayer membrane: buffer partition coefficient of pentobarbitone on pH and lipid composition.

1 The membrane/buffer partition coefficient of [14C]-pentobarbitone has been determined as a function of the lipid composition of bilayer membranes. 2 A new technique based on ultrafiltration gave comparable results to conventional techniques but required less time for equilbration. 3 The membrane/buffer coefficient was independent of pentobarbitone concentration in the range studies. 4 The apparent partition coefficient varied with pH and was a linear function of the degree of dissociation of pentobarbition. 5 Both the charged and uncharged forms of pentobarbitone partitioned into the membrane, the latter to a much greater extent than the former. 6 At low pH the highest partition coefficient observed was in egg phosphatidylcholine bilayer membranes. 7 Incorporation of cholesterol or phosphatidic acid into phosphatidylcholine membranes greatly reduced the partition coefficient. 8 High pressures do not greatly change these partition coefficients.     

First-principles, structure-based transdermal transport model to evaluate lipid partition and diffusion coefficients of hydrophobic permeants solely from stratum corneum permeation experiments

To account for the effect of branched, parallel transport pathways in the intercellular domain of the stratum corneum (SC) on the passive transdermal transport of hydrophobic permeants, we have developed, from first-principles, a new theoretical model-the Two-Tortuosity Model. This new model requires two tortuosity factors to account for: (1) the effective diffusion path length, and (2) the total volume of the branched, parallel transport pathways present in the SC intercellular domain, both of which may be evaluated from known values of the SC structure. After validating the Two-Tortuosity model with simulated SC diffusion experiments in FEMLAB (a finite element software package), the vehicle-bilayer partition coefficient, K(b), and the lipid bilayer diffusion coefficient, D(b), in untreated human SC were evaluated using this new model for two hydrophobic permeants, naphthol (K(b) = 225 +/- 42, D(b) = 1.7 x 10(-7) +/- 0.3 x 10(-7) cm(2)/s) and testosterone (K(b) = 92 +/- 29, D(b) = 1.9 x 10(-8) +/- 0.5 x 10(-8) cm(2)/s). The results presented in this paper demonstrate that this new method to evaluate K(b) and D(b) is comparable to, and simpler than, previous methods, in which SC permeation experiments were combined with octanol-water partition experiments, or with SC solute release experiments, to evaluate K(b) and D(b).     

High-performance liquid chromatographic analysis of the investigational anticancer drug 9-aminocamptothecin, as the lactone form and as the total of the lactone and the hydroxycarboxylate forms, in micro-volumes of human plasma

A high performance liquid chromatographic (HPLC) assay is described for the determination of the investigational anticancer drug 9 aminocamptothecin (9-AC) as the lactone form (9AC(lac)) and as the total of the lactone and hydroxycarboxylate forms (9AC-(tot)), in micro volumes of plasma. The analytical methodology reported here involves a protein precipitation step with cold methanol (−30°C) as sample pretreatment procedure. The methanolic extract is used for the determination of 9AC-(tot). The intact (active) lactone form of 9-AC is separated from the hydroxycarboxylate form in the methanolic plasma extract by solid phase extraction within 48 h after sampling and deproteination. After evaporation to dryness (nitrogen, 40°C) the extract can be stored at −70°C for at least 3 weeks. The drug is analysed by reversed-phase liquid chromatography on a Zorbax SB RP-18 column, using methanol–water eluent (pH 2.2) and fluorescence detection. The presented assay is linear over a concentration range 0.2–100 ng·ml⁻¹ with a detection limit and a limit of quantitation of 0.05 and 0.2 ng·ml⁻¹, respectively, for both 9-AC(tot) and 9-AC(lac) using a 100 ml plasma sample. The proposed method has been implemented in a phase I clinical trial for pharmacokinetic evaluation of this potential new drug.     

Induced desensitization of the insulinotropic effects of antidiabetic drugs, BTS 67 582 and tolbutamide

Acute and chronic mechanisms of action of novel insulinotropic antidiabetic drug, BTS 67 582 (1, 1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate), were examined in the stable cultured BRIN-BD11 cell line. BTS 67 582 (100 - 400 microM) stimulated a concentration-dependent increase (P<0.01) in insulin release at both non-stimulatory (1.1 mM) and stimulatory (8. 4 mM) glucose. Long-term exposure (3 - 18 h) to 100 microM BTS 67 582 in culture time-dependently decreased subsequent responsiveness to acute challenge with 200 microM BTS 67 582 or 200 microM tolbutamide at 12 - 18 h (P<0.001). Similarly 3 - 18 h culture with the sulphonylurea, tolbutamide (100 microM), also effectively suppressed subsequent insulinotropic responses to both BTS 67 582 and tolbutamide. Culture with 100 microM BTS 67 582 or 100 microM tolbutamide did not affect basal insulin secretion, cellular insulin content, or cell viability and exerted no influence on the secretory responsiveness to 200 microM of the imidazoline, efaroxan. While 18 h BTS 67 582 culture did not affect the insulin-releasing actions (P<0.001) of 16.7 mM glucose, 10 mM arginine, 30 mM KCl, 25 microM forskolin or 10 nM phorbol-12-myristate 13-acetate (PMA), significant inhibition (P<0.001) of the insulinotropic effects of 10 mM 2-ketoisocaproic acid (KIC) and 10 mM alanine were observed. These data suggest that BTS 67 582 shares a common signalling pathway to sulphonylurea but not imidazoline drugs. Desensitization of drug action may provide an important approach to dissect sites of action of novel and established insulinotropic antidiabetic agents.     

Effect of lipid bilayer alteration on transdermal delivery of a high-molecular-weight and lipophilic drug: studies with paclitaxel

Skin forms an excellent barrier against drug permeation, due to the rigid lamellar structure of the stratum corneum (SC) lipids. Poor permeability of drugs can be enhanced through alteration in partition and diffusion coefficients, or concentration gradient of drug with an appropriate choice of solvent system, along with penetration enhancers. The aim of the current investigation was to assess applicability of lipid bilayer alteration by fatty acids and terpenes toward the permeation enhancement of a high-molecular-weight, lipophilic drug, paclitaxel (PCL) through rat skin. From among the fatty acids studied using ethanol/isopropyl myristate (1:1) vehicle, no significant enhancement in flux of PCL was observed (p > 0.05). In the case of cis mono and polyunsaturated fatty acids lag time was found to be similar to control (p > 0.05). This suggests that the permeation of a high-molecular-weight, lipophilic drug may not be enhanced by the alteration of the lipid bilayer, or the main barrier to permeation could lie in lower hydrophilic layers of skin. A significant increase in lag time was observed with trans unsaturated fatty acids unlike the cis isomers, and this was explained on the basis of conformation and preferential partitioning of fatty acids into skin. From among the terpenes, flux of PCL with cineole was significantly different from other studied terpenes and controls, and after treatment with menthol and menthone permeability was found to be reduced. Menthol and menthone cause loosening of the SC lipid bilayer due to breaking of hydrogen bonding between ceramides, resulting in penetration of water into the lipids of the SC lipid bilayer that leads to creation of new aqueous channels and is responsible for increased hydrophilicity of SC. This increased hydrophilicity of the SC bilayer might have resulted in unfavorable conditions for ethanol/isopropyl myristate (1:1) along with PCL to penetrate into skin, therefore permeability was reduced. The findings of this study suggest that the permeation of a high-molecular-weight and lipophilic drug cannot be enhanced through bilayer alteration by penetration enhancers, and alteration in partitioning of drug into skin could be a feasible mode to enhance the permeation of drug.     

Pharmacokinetic interplay of phase II metabolism and transport: a theoretical study

Understanding of the interdependence of cytochrome P450 enzymes and P-glycoprotein in disposition of drugs (also termed transport-metabolism interplay) has been significantly advanced in recent years. However, whether such interplay exists between phase II metabolic enzymes and efflux transporters remains largely unknown. The objective of this article is to explore the role of efflux transporters (acting on the phase II metabolites) in disposition of the parent drug in Caco-2 cells, liver, and intestine via simulations utilizing a catenary model (for Caco-2 system) and physiologically based pharmacokinetic (PBPK) models (for the liver and intestine). In all three models, transport-metabolism interplay (i.e., inhibition of metabolite efflux decreases the metabolism) can be observed only when futile recycling (or deconjugation) occurred. Futile recycling appeared to bridge the two processes (i.e., metabolite formation and excretion) and enable the interplay thereof. Without futile recycling, metabolite formation was independent on its downstream process excretion, thus impact of metabolite excretion on its formation was impossible. Moreover, in liver PBPK model with futile recycling, impact of biliary metabolite excretion on the exposure of parent drug [(systemic (reservoir) area under the concentration-time curve (AUC(R1))] was limited; a complete inhibition of efflux resulted in AUC(R1) increases of less than 1-fold only. In intestine PBPK model with futile recycling, even though a complete inhibition of efflux could result in large elevations (e.g., 3.5-6.0-fold) in AUC(R1), an incomplete inhibition of efflux (e.g., with a residual activity of ≥ 20% metabolic clearance) saw negligible increases (<0.9-fold) in AUC(R1). In conclusion, this study presented mechanistic observations of pharmacokinetic interplay between phase II enzymes and efflux transporters. Those studying such interplay are encouraged to adequately consider potential consequences of inhibition of efflux transporters in humans.