Evaluation and prediction of pharmacokinetics of PFOA and PFOS in the monkey and human using a PBPK model

Perfluoroalkyl acid carboxylates and sulfonates (PFAAs) have many consumer and industrial applications. The persistence and widespread distribution of these compounds in humans have brought them under intense scrutiny. Limited pharmacokinetic data is available in humans; however, human data exists for two communities with drinking water contaminated by PFAAs. Also, there is toxicological and pharmacokinetic data for monkeys, which can be quite useful for cross-species extrapolation to humans. The goal of this research was to develop a physiologically-based pharmacokinetic (PBPK) model for PFOA and PFOS for monkeys and then scale this model to humans in order to describe available human drinking water data. The monkey model simulations were consistent with available PK data for monkeys. The monkey model was then extrapolated to the human and then used to successfully simulate the data collected from residents of two communities exposed to PFOA in drinking water. Human PFOS data is minimal; however, using the half-life estimated from occupational exposure, our model exhibits reasonable agreement with the available human serum PFOS data. It is envisioned that our PBPK model will be useful in supporting human health risk assessments for PFOA and PFOS by aiding in understanding of human pharmacokinetics.     

A unified algorithm for predicting partition coefficients for PBPK modeling of drugs and environmental chemicals

The algorithms in the literature focusing to predict tissue:blood PC (P_tb) for environmental chemicals and tissue:plasma PC based on total (K_p) or unbound concentration (K_pu) for drugs differ in their consideration of binding to hemoglobin, plasma proteins and charged phospholipids. The objective of the present study was to develop a unified algorithm such that P_tb, K_p and K_pu for both drugs and environmental chemicals could be predicted. The development of the unified algorithm was accomplished by integrating all mechanistic algorithms previously published to compute the PCs. Furthermore, the algorithm was structured in such a way as to facilitate predictions of the distribution of organic compounds at the macro (i.e. whole tissue) and micro (i.e. cells and fluids) levels. The resulting unified algorithm was applied to compute the rat P_tb, K_p or K_pu of muscle (n = 174), liver (n = 139) and adipose tissue (n = 141) for acidic, neutral, zwitterionic and basic drugs as well as ketones, acetate esters, alcohols, aliphatic hydrocarbons, aromatic hydrocarbons and ethers. The unified algorithm reproduced adequately the values predicted previously by the published algorithms for a total of 142 drugs and chemicals. The sensitivity analysis demonstrated the relative importance of the various compound properties reflective of specific mechanistic determinants relevant to prediction of PC values of drugs and environmental chemicals. Overall, the present unified algorithm uniquely facilitates the computation of macro and micro level PCs for developing organ and cellular-level PBPK models for both chemicals and drugs.     

Targeted delivery of a poorly water-soluble compound to hair follicles using polymeric nanoparticle suspensions

This study explored the utility of topically applied polymeric nanoparticle suspensions to target delivery of poorly water-soluble drugs to hair follicles. Several formulations of amorphous drug/polymer nanoparticles were prepared from ethyl cellulose and UK-157,147 (systematic name (3S,4R)-[6-(3-hydroxyphenyl)sulfonyl]-2,2,3-trimethyl-4-(2-methyl-3-oxo-2,3-dihydropyridazin-6-yloxy)-3-chromanol), a potassium channel opener, using sodium glycocholate (NaGC) as a surface stabilizer. Nanoparticle suspensions were evaluated to determine if targeted drug delivery to sebaceous glands and hair follicles could be achieved. In in vitro testing with rabbit ear tissue, delivery of UK-157,147 to the follicles was demonstrated with limited distribution to the surrounding dermis. Delivery to hair follicles was also demonstrated in vivo, based on stimulation of hair growth in tests of 100-nm nanoparticles with a C3H mouse model. The nanoparticles were well-tolerated, with no visible skin irritation. In vivo tests of smaller nanoparticles with a hamster ear model also indicated targeted delivery to sebaceous glands. The nanoparticles released drug rapidly in in vitro nonsink dissolution tests and were stable in suspension for 3 months.The present results show selective drug delivery to the follicle by follicular transport of nanoparticles and rapid release of a poorly water-soluble drug. Thus, nanoparticles represent a promising approach for targeted topical delivery of low-solubility compounds to hair follicles.     

Physiologically based pharmacokinetic modelling and in vivo [I]/Ki accurately predict P-glycoproteinmediated drug-drug interactions with dabigatran etexilate

Background and purpose

In vitro inhibitory potency (K_i)-based predictions of P-glycoprotein (P-gp)-mediated drug-drug interactions (DDIs) are hampered by the substantial variability in inhibitory potency. In this study, in vivo-based [I]/K_i values were used to predict the DDI risks of a P-gp substrate dabigatran etexilate (DABE) using physiologically based pharmacokinetic (PBPK) modelling.

Experimental approach

A baseline PBPK model was established with digoxin, a known P-gp substrate. The K_m (P-gp transport) of digoxin in the baseline PBPK model was adjusted to K_mⁱ to fit the change of digoxin pharmacokinetics in the presence of a P-gp inhibitor. Then ‘in vivo’ [I]/K_i of this P-gp inhibitor was calculated using K_mⁱ/K_m. Baseline PBPK model was developed for DABE, and the ‘in vivo’ [I]/K_i was incorporated into this model to simulate the static effect of P-gp inhibitor on DABE pharmacokinetics. This approach was verified by comparing the observed and the simulated DABE pharmacokinetics in the presence of five different P-gp inhibitors.

Key results

This approach accurately predicted the effects of five P-gp inhibitors on DABE pharmacokinetics (98–133% and 89–104% for the ratios of AUC and C_max respectively). The effects of 16 other P-gp inhibitors on the pharmacokinetics of DABE were also confidently simulated.

Conclusions and implications

‘In vivo’ [I]/K_i and PBPK modelling, used in combination, can accurately predict P-gp-mediated DDIs. The described framework provides a mechanistic basis for the proper design of clinical DDI studies, as well as avoiding unnecessary clinical DDI studies.     

Application of unbound liver-to-plasma concentration ratio to quantitative projection of cytochrome P450-mediated drug–drug interactions using physiologically based pharmacokinetic modelling approach

Abstract

1. This study evaluated the prediction accuracy of cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) using minimal physiologically-based pharmacokinetic (PBPK) modelling incorporating the hepatic accumulation factor of an inhibitor (i.e. unbound liver/unbound plasma concentration ratio [K_p,uu,liver]) based on 22 clinical DDI studies.

2. K_p,uu,liver values were estimated using three methods: (1) ratio of cell-to-medium ratio in human cryopreserved hepatocytes (C/M_u) at 37?°C to that on ice (K_p,uu,C/M), (2) multiplication of total liver/unbound plasma concentration ratio (K_p,u,liver) estimated from C/M_u at 37?°C with unbound fraction in human liver homogenate (K_p,uu,cell) and (3) observed K_p,uu,liver in rats after intravenous infusion (K_p,uu,rat).

3. PBPK model using each K_p,uu,liver projected the area under the curve (AUC) increase of substrates more accurately than the model assuming a K_p,uu,liver of 1 for the average fold error and root mean square error did. Particularly, the model with a K_p,uu,liver of 1 underestimated the AUC increase of triazolam following co-administration with CYP3A4 inhibitor itraconazole by five-fold, whereas the AUC increase projected using the model incorporating the K_p,uu,C/M, K_p,uu,cell, or K_p,uu,rat of itraconazole and hydroxyitraconazole was within approximately two-fold of the actual value.

4. The results indicated that incorporating K_p,uu,liver into the PBPK model improved the accuracy of DDI projection.     

Using population physiologically based pharmacokinetic modeling to determine optimal sampling times and to interpret biological exposure markers: The example of occupational exposure to styrene

Background

Biomonitoring of chemicals in the workplace provides an integrated characterization of exposure that accounts for uptake through multiple pathways and physiological parameters influencing the toxicokinetics.

Objectives

We used the case of styrene to (i) determine the best times to sample venous blood and end-exhaled air, (ii) characterize the inter-individual variability in biological levels following occupational exposure and (iii) propose biological limit values using a population physiologically based pharmacokinetic (PBPK) model.

Methods

We performed Monte Carlo simulations with various physiological, exposure and workload scenarios. Optimal sampling times were identified through regression analyses between levels in biological samples and 24-h area under the arterial blood concentration vs. time curve. We characterized the variability in levels of styrene in biological samples for exposures to a time weighted average (TWA) of 20 ppm.

Results

Simulations suggest that the best times to sample venous blood are at the end of shift in poorly ventilated workplaces and 15 min after the shift in highly ventilated workplaces. Exhaled air samples are most informative 15 min after the shift. For a light workload, simulated styrene levels have a median (5th–95th percentiles) of 0.4 mg/l (0.2–0.6) in venous blood at the end of shift and 0.5 ppm (0.3–0.8) in exhaled air 15 min after the end of shift.

Conclusion

This study supports the current BEI^® of the ACGIH of 0.2 mg/l of styrene in venous blood at the end of shift and indicates a biological limit value of 0.3 ppm in end-exhaled air 15 min after the end of shift.     

Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation

Trivalent methylated metabolites of arsenic, monomethylarsonous acid (MMA^III) and dimethylarsinous acid (DMA^III), have been found highly reactive and toxic in various cells and in vivo animal models, suggesting their roles in the arsenic-associated toxicity. However, their effects on cardiovascular system including blood cells, one of the most important targets for arsenic toxicity, remain poorly understood. Here we found that MMA^III and DMA^III could induce procoagulant activity and apoptosis in platelets, which play key roles in the development of various cardiovascular diseases (CVDs) through excessive thrombus formation. In freshly isolated human platelets, treatment of MMA^III resulted in phosphatidylserine (PS) exposure, a hallmark of procoagulant activation, accompanied by distinctive apoptotic features including mitochondrial membrane potential disruption, cytochrome c release, and caspase-3 activation. These procoagulant activation and apoptotic features were found to be mediated by the depletion of protein thiol and intracellular ATP, and flippase inhibition by MMA^III, while the intracellular calcium increase or reactive oxygen species generation was not involved. Importantly, increased platelet procoagulant activity by MMA^III resulted in enhanced blood coagulation and excessive thrombus formation in a rat in vivo venous thrombosis model. DMA^III also induced PS-exposure with apoptotic features mediated by protein thiol depletion, which resulted in enhanced thrombin generation. In summary, we believe that this study provides an important evidence for the role of trivalent methylated arsenic metabolites in arsenic-associated CVDs, giving a novel insight into the role of platelet apoptosis in toxicant-induced cardiovascular toxicity.     

Antagonist inhibition curves and the measurement of dissociation constants

1. Experiments carried out on guinea-pig isolated ileum with carbachol as agonist and diphenyl- acetoxyethyl- dimethyl-ethyl- ammonium (DADMEA) bromide as antagonist gave results which fit the theoretical relation between fractional inhibition (Q) of the effects of an agonist ([A]) and the concentration of a competitive antagonist ([B]): this also involves the Hill coefficient (logistic slope factor, P) for the agonist concentration-response curve and the degree of agonist stimulation, [A]/[A]₅₀, where [A]₅₀ produces a half-maximum response.
2. Values of IC₅₀ and an exponent, P′, can be obtained by fitting Q to [B] using a logistic approximation to the relation. Both P′ and IC₅₀ should be greater with higher agonist stimulation but the increase in P′ may be masked by errors in extreme values of Q. Estimates of IC₅₀, however, invariably increased with higher agonist stimulation but with a steep concentration-response curve (P>1) and low agonist stimulation ([A]/[A]₅₀ <1), IC₅₀ can be less than K_D.
3. K_D was calculated from the results in three ways: (i) by a least-squares fit of Q to [B] using the values of P and [A]/[A]₅₀ calculated from the control concentration-response curve; (ii) from the value of IC₅₀ for each line and the values of P and [A]/[A]₅₀ and (iii) by using the agonist concentration-response curve to calculate the dose-ratio and estimate of K_D for each response in the presence of the antagonist. The methods gave similar results (nM: 11 experiments), 12.4±1.1 (i), 11.7±0.9 (ii), 14.8±1.6 (iii) but there are advantages in using methods (i) or (ii) rather than (iii).
4. The method by which K_D is calculated is less important than the experimental design: the plan used in this work, with alternative small and large responses from the tissue, is very suitable for estimating K_D with low concentrations of antagonists and small dose-ratios. Although it is not a sensitive test for competitive behaviour because only a small range of concentrations of antagonist is tested, the estimate of affinity should be free from complications involved in the use of higher concentrations of antagonist (and agonist) and the nature of the antagonism can always be checked by doing further experiments in the presence of a known competitive antagonist.

     

Strategies to improve efficacy and safety of a novel class of antiviral hyper-activation-limiting therapeutic agents: the VS411 model HIV/AIDS

BACKGROUND AND PURPOSE

Antiviral hyper-activation-limiting therapeutic agents (AV-HALTs) are a novel experimental drug class designed to both decrease viral replication and down-regulate excessive immune system activation for the treatment of chronic infections, including human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome. VS411, a first-in-class AV-HALT, is a single-dosage form combining didanosine (ddI, 400 mg), an antiviral (AV), and hydroxyurea (HU, 600 mg), a cytostatic agent, designed to provide a slow release of ddI to reduce its maximal plasma concentration (C_max) to potentially reduce toxicity while maintaining total daily exposure (AUC) and the AV activity.

EXPERIMENTAL APPROACH

This was a pilot phase I, open-label, randomized, single-dose, four-way crossover trial to investigate the fasted and non-fasted residual variance of AUC, C_max and the oral bioavailability of ddI and HU, co-formulated as VS411, and administered as two different fixed-dose combination formulations compared to commercially available ddI (Videx EC) and HU (Hydrea) when given simultaneously.

KEY RESULTS

Formulation VS411-2 had a favourable safety profile, displayed a clear trend for lower ddI C_max (P = 0.0603) compared to Videx EC, and the 90% confidence intervals around the least square means ratio of C_max did not include 100%. ddI AUC_∞ was not significantly decreased compared to Videx EC. HU pharmacokinetic parameters were essentially identical to Hydrea, although there was a decrease in HU exposure under fed versus fasted conditions.

CONCLUSIONS AND IMPLICATIONS

A phase IIa trial utilizing VS411-2 formulation has been fielded to identify the optimal doses of HU plus ddI as an AV-HALT for the treatment of HIV disease.     

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE

KCNQ-encoded voltage-gated potassium channels (K_v7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

EXPERIMENTAL APPROACH

Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K_v7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels.

KEY RESULTS

KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K_v7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K_v7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K⁺ currents. In contrast, the K_v7.1-specific blocker chromanol 293B did not affect vascular tone. Two K_v7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K_v7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991.

CONCLUSIONS AND IMPLICATIONS

This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo.

LINKED ARTICLE

This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x     

Evaluation of bisphenol A glucuronidation according to UGT1A1*28 polymorphism by a new LC-MS/MS assay

The endocrine disruptor bisphenol A (BPA) is a frequently used chemical in the manufacture of consumer products. In humans, BPA is extensively metabolized to BPA glucuronide (BPAG) by different UDP-glucuronosyltransferase (UGT) isoforms. The study has been performed with the intention to improve the accuracy of published physiologically based pharmacokinetic models and to improve regulatory risk assessments of BPA. In order to gain insight into intestine, kidney, liver, and lung glucuronidation of BPA, human microsomes of all tested organs were used. BPAG formation followed Michaelis-Menten kinetics in the intestine and kidney, but followed substrate inhibition kinetics in the liver. Human lung microsomes did not show glucuronidation activity towards BPA. While the liver intrinsic clearance was very high (857 mL min(-1)kg body weight(-1)), the tissue intrinsic clearances for the kidney and intestine were less than 1% of liver intrinsic clearance. Since BPA is a UGT1A1 substrate, we postulated that the common UGT1A1*28 polymorphism influences BPA glucuronidation, and consequently, BPA detoxification. Hepatic tissue intrinsic clearances for UGT1A1*1/*1, UGT1A1*1/*28, and UGT1A1*28/*28 microsomes were 1113, 1075, and 284 mL min(-1)kg body weight(-1), respectively. Prior to microsomal experiments, the bioproduction of BPAG and stable isotope-labeled BPAG (BPAG(d16)) was performed for the purpose of the reliable and accurate quantification of BPAG. In addition, a sensitive LC-MS/MS analytical method for the simultaneous determination of BPA and BPAG based on two stable isotope-labeled internal standards was developed and validated. In conclusion, our in vitro results show that the liver is the main site of BPA glucuronidation (K(m) 8.9 μM, V(max) 8.5 nmol min(-1) mg(-1)) and BPA metabolism may be significantly influenced by a person's genotype (K(m) 10.0-13.1 μM, V(max) 3.4-16.2 nmol min(-1) mg(-1)). This discovery may be an important fact for the currently on-going worldwide BPA risk assessments and for the improvement of physiologically based pharmacokinetic models.     

Expression of proliferative and inflammatory markers in a full-thickness human skin equivalent following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 μM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 μM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE₂ synthases, leukotriene (LT) A₄ hydrolase and LTC₄ synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.     

Evaluation of an eucalyptus oil containing topical drug delivery system for selected steroid hormones

In the present study the permeation and the chemical stability of 17-beta-estradiol, progesterone, cyproterone acetate and finasteride incorporated in an eucalyptus oil containing microemulsion system have been investigated. The formulations contained 1% (w/w) of the steroid hormones. Self diffusion coefficients determined by pulsed-field-gradient spin echo NMR spectroscopy were used to characterise the microemulsion. From these results a bicontinuous structure is proposed for the multicomponent system. However a correlation between the self diffusion of the hormones in the vehicle and the transdermal flux was not indicated. Explanations for this were self assembling, formation of aggregates between the components of the microemulsion and drugs and different effects because of different solubility of the drugs. By addition of certain polymers the skin permeation rates could be improved with exception of cyproterone acetate. Beside standard diffusion experiments, the residual drug content in the skin was investigated. Drug stability was monitored by analysing the steroid hormone content in the different formulations over an observation period of 6 weeks and could be improved by polymers. In addition, viscosity measurements were performed. They indicated an influence of the polymers and drugs on the viscosity in all formulations.     

Sensory and pulmonary effects of acute exposure to sulfur dioxide (SO2)

Background

Exposure to sulfur dioxide (SO₂) affects large populations worldwide. Pulmonary effects have been reported at concentrations relevant in the general (<0.5 ppm) and working environment (>0.5 ppm). SO₂ is an irritant but the existing studies often emphasize only pulmonary effects and no clear dose–response relationship has yet been described.

Objectives

Using a multi-level, multi-method approach, odor annoyance, sensory irritation and pulmonary effects of SO₂ were to be investigated in an experimental exposure study.

Methods

Eye blink frequency, rhinomanometry, spirometry and symptom ratings of acute health effects were assessed before, during, and after the exposures. Each session lasted 4 h and concentrations of 0.5, 1, and 2 ppm were investigated and compared to a control condition using clean air. Sixteen human volunteers (8 females/8 males) participated and during exposure light physical exercise was simulated with bicycle ergometry.

Results

Eye blink frequency, nasal airflow, and lung function were not affected by the acute SO₂ exposure investigated. These physiological responses to moderate SO₂ exposures were not significantly affected by gender. Regarding subjectively measured chemosensory sensations, only odor annoyance ratings increased in a dose-dependent manner, but the average magnitudes were labeled weak to moderate.

Conclusions

In healthy volunteers, without hyperresponsiveness to SO₂, no dose-dependent effects of acute SO₂ exposures up to 2 ppm could be measured. Due to olfactory perceptions subjects were aware of the different SO₂ exposures but the associated annoyance was relatively low.     

Effects of polyphenols derived from fruit of Crataegus pinnatifida on cell transformation, dermal edema and skin tumor formation by phorbol ester application.

The dried fruits of Crataegus pinnatifida have been used traditionally as oriental medicine and local soft drink material recently. Previously, we demonstrated that C. pinnatifida exhibited anti-oxidation and anti-inflammatory potential. To clarify the active components in anti-transformation and anti-tumor promotion, we collected the polyphenol fraction (CF-TP) of hot-water extracts from dried fruits of C. pinnatifida for the following study. By anchorage-independent transformation assay, CF-TP significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation in JB6 P(+) cells. Moreover, we found that CF-TP inhibited the expression of osteopontin (OPN), a transformational marker, and the activation of NF-kappaB and AP-1 induced by TPA in JB6 P(+) cells. In addition, we evaluated the effect of CF-TP on TPA application to ICR mouse skin with measurement of H(2)O(2) production, myeloperoxidase (MPO) activity, edema formation, epidermal thickness and leukocyte infiltration. As a result, CF-TP significantly inhibited the generation of reactive oxygen species (ROS) and the phenomena of inflammation induced by TPA. It also suppressed the expression of COX-2 and iNOS, and the activation of ornithine decarboxylase (ODC). Furthermore, CF-TP inhibited benzo[a]pyrene (B[a]P)/TPA-induced skin tumor formation and decreased the incidence of tumor. These results indicate that CF-TP possesses potential as a cancer chemopreventive agent against tumor promotion.     

Ring Structure and Aromatic Substituent Effects on the pKa of the Benzoxaborole Pharmacophore

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The modulation by xanthines of the DNA-damaging effect of polycyclic aromatic agents. Part II. The stacking complexes of caffeine with doxorubicin and mitoxantrone

Recently accumulated statistical data indicate the protective effect of caffeine consumption against several types of cancer diseases. There are also reports about protective effect of caffeine and other xanthines against tumors induced by polycyclic aromatic hydrocarbons. One of the explanations of this phenomenon is based on biological activation of such carcinogens by cytochromes that are also known for metabolism of caffeine. In the accompanying paper [Kapuscinski et al., this issue] we provide evidence (flow cytometry and the cell cycle analysis) that the cytostatic effects of caffeine (CAF) on two DNA alkylating agents, which do not require the biological activation, depend on their ability to form stacking (pi-pi) complexes. In this study, we use physicochemical techniques (computer aided light absorption and microcalorimetry), and molecular modeling to examine previously published qualitative data. This is published both by our and other group's data, indicates that CAF is able to modify the cytotoxic and/or cytostatic action of the two well known antitumor drugs doxorubicin (DOX) and mitoxantrone (MIT). To obtain the quantitative results from the experimental data we used the statistical-thermodynamical model of mixed aggregation, to find the association constants K(AC) of the CAF-drug interaction (128+/-10 and 356+/-21M(-1) for DOX-CAF and MIT-CAF complex formation, respectively). In addition, the favorable enthalpy change of CAF-MIT (DeltaH=-11.3kcal/mol) was measured by microcalorimetry titration. The molecular modeling (semi-empirical and force field method) allowed us to obtain the geometry of these complexes, which indicated the favorable energy (DeltaE) of complex formation of the protonated drug's molecules in aqueous environment (-7.4 and -8.7kcal/mol for DOX-CAF.5H(2)O and MIT-CAF.8H(2)O complex, respectively). The molecular modeling calculation indicates the existence of CAF-drug complexes in which the MIT molecules are intercalated between two CAF molecules (DeltaE=-29.9kcal/mol). These results indicate that the attenuating effect of caffeine on cytotoxic or mutagenic effects of some polycyclic aromatic mutagens cannot be the result of metabolic activation in the cells, but simply is the physicochemical process of the sequestering of aromatic molecules (e.g. carcinogens or mutagens) by formation of the stacking complexes. The caffeine may then act as the "interceptor" of potential carcinogens (especially in the upper part of digesting track) where its concentration can reach the mM level). There is, however, no indication, both, in the literature or from our experiments, that the xanthines can reverse the damage to nucleic acids at the point when the damage to DNA has already occurred.