UMF - Universitatea de Medicina si Farmacie "Carol Davila" Bucuresti

The study of the effects of biological matrices interactions with some food on the variability of bioanalystic results

CNMP - PN II contract - Partnerships in priority areas

Thematic area: IV - Health

Contract number: 42-100/2008

Acronym: BIOMARTIX

Project Director: Prof. Dr. Maria Crina Monciu

Project duration: 36 months (2008-2011)

Contract value: 2250000 lei (2000000 of the budget, 250,000 of co)

The partners involved in this project are:

CO: University of Medicine and Pharmacy "Carol Davila" (UMFCD)

P1: University of Bucharest (UB)

P2: SC Biopharmacy & Pharmacol RES SA (BIOPHARM)

Summary:

The project aims to investigate the effect of human plasma composition changes after the absorption of food on the results of extraction methods and chromatographic liquid dosage in pharmacokinetic studies. This effect will be referred to as "matrixfood effect". Such a research is required primarily in the methods applied in bioavailability and bioequivalence studies, because these studies usually lasts over 24 hours and samples corresponding to two very different physiological conditions: those after a period of at least 10 hours of fasting and those after the standard meals served to the subjects.

The research that we will perform will also take into consideration the fact that for absorption of food, the body uses as absorption promoters salts of the bile acids due to their tensioctive properties and the ability to carry digestion products. These salts have effects at the same time on the absorption, distribution and elimination of drugs.

From the preliminary investigations that we conducted, we found that these effects, alone or in combination, cause a high variability of results obtained when applying separation and cromatographic analysis methods, with negative consequences on the process of validation of these methods. There will be studied the effects of standard food composition used in clinical studies, the tensioactive physiological effects, and the effects of nanostructured systems - as a model simulating the combined effect of the two components. At the same time as physiological surfactants have a very high cost and non-unitary composition, will be considered alternative mixtures of synthetic surfactants, much easier to standardize. Correlations between the obtained results and the test of the predictive power of the  alternative methods will be done with advanced methods of biostatistics.

The project team includes the departments of analytical chemistry, biostatistics, colloidal chemistry, pharmacology, and medical specialists in gastroenterology and laboratory medicine, and a SME that actually applies such types of methods in clinical pharmacokinetic studies.

Objectives:

Main objective:

The project aims to separate the components of the bioanalytical variability results in order to emphasize the factor generated by the interaction of drug-food, which has not been considered so far and that may have, at some point, in the pharmacokinetic studies longer than several hours, an influence far greater than all other factors of variability.

Specific objectives.

   1. Estimating in vivo changes by comparing plasma samples collected in pharmacokinetic studies before meal with those after meal.
   2. Since the absorption of foods, especially the lipids is made using physiologic surfactants, bile acids and salts, it was proposed to study the influence of these agents on the analytical results at different concentrations, especially near the critical micellar concentration and the concentrations close to those estimated in postprandial blood samples.
   3. In vitro creation of dispersed nanostructured systems to simulate the total composition of postprandial plasma.
   4. Replacing the physiological surfactants, bile acids and salts with synthetic surfactants, which allow an alternative method of investigation of their effects
   5. The ultimate goal: developing a methodology for validation of bioanalytical methods to also include in the "robust" chapter, the stability of method parameters at plasma composition changes by simulating changes induced by the absorption of food. 

Workplan

Budget

Results:

Phase I / 2009:

Summary

Activity I.1 (CO, P2)

Development of methods for separation and simultaneous dosing for lipophilic drugs and their active metabolites (ketoconazole, omeprazole, glyburide, carbamazepine and epoxicarbamazepine, nimesulid and hydroxy - nimesulid, itraconazole and hydroxy-itraconazole)

Results: Have been developed simultaneously dosing methods in plasma for the active substances considered in the study and metabolites. The methods have been validated and can be applied in studies of experimental and clinical pharmacokinetics.

Activity I.2 (P2)

Qualitative Estimation of the effects due to plasma composition on the in vivo analytical results, by comparing data obtained in clinical studies before and after standard meals administration.

Results: The phenomenological and statistical analysis of about 1500 measurements regarding the concentration of an internal standard (lansoprazole) in a bioequivalence study of two pharmaceutical formulations based of omeprazole have identified two phenomena: 1. correlations between measured areas and time on intervals of less than 12 h, correlations that no longer appear when the range is increased at 24 h; 2. appearance of a local minimum around the standard meal received by the subjects after 12 hours of fasting.

Activity I.3 (P1, P2)

Analysis and modeling of transfer phenomena in order to separate the effects on extraction of loss of analyte effects or interference effects with food compounds or physiological compounds associated with food absorption

Results. Were evaluated experimental methods and theoretical models of transfer phenomena in colloidal systems, methods and models to be subsequently applied in complex systems including food components, medicinal active substances and their metabolites. Experimental studies were conducted:

- regarding the possible transfer mechanisms of two homogeneous phases (simulating plasma and solvent extraction) and heterogeneous phase (micelles and microemulsions or nanoemulsions) in the presence of the model systems.

- regarding the methods for investigating the transfer of the analyte in and from colloidal systems,

- regarding the influence of the dispersed phase drops size on the kinetics of analyte transfer from microemulsion.

 - was performed the methodology to investigate the transfer of some model analytes with hydrophilic and hydrophobic characteristics in systems containing SDS micelles and microemulsions.

Activity I.4 (CO)

Defining a global quantitative parameter "Matrix postprandial factor (FMPP)" to characterize the effect of food on analytical results:

FMMP = the post-prandial peak area / the pre-prandial peak area

or the ratio of normalized areas in relation to internal standard area. Analysis of the concept consistency. The search of other quantitative parameters to measure post-prandial matrix effects.

Results. Were compared the peak areas of lansoprazole near the minimum standard associated with the standard meal after 12 h of fasting and 4 h from the drug administration. In fact there was no jump at a specific time, so as it was expected in the project draft. The phenomenon seems to be more complex associated with the interference of food compounds on the extraction process but also with a periodic evolution of physiological conditions, in fact the composition of plasma, depending on the pre- or postprandial status. In fact there is a continuing increase of the peaks area in conditions of fasting, the trend being reversed for a while after food absorption.

We may say that the appearance of food compounds in the plasma leads to a decrease of 10-20% of the peak-area but reducing food interference with the processes of transfer of only this phenomenon and ignoring some associated physiological phenomena is a simplified model. It is going to find ways and models of the separation of the two phenomena.

Phase II/2009

SUMMARY

The concrete scientific objectives of the phase were related to the verification of the following working hypotheses based on previous results of the authors and the results obtained in the previous phase.

   1. Extraction yield effectively depends on the concentration of surfactant
   2. The dependency of the effect of the surfactant concentration changes depending on the concentration of the analyte
   3. In the area of the surfactant critical micellar concentration large fluctuations occur and even reversals of the effects.
   4. Beyond the critical micellar concentration yield increases with the increasing of the concentration of surfactant.

 Experimental methods

Extraction kinetics and yield of the plasma depending on the nature and concentration of synthetic surfactants in the field of the concentrations below and above critical micellar concentration were studied in the case of two poorly soluble substances: carbamazepine and ketoconazole.

It was used as surfactant Tween-80 in concentrations in the range 0 to 0.1% (0, 0.005, 0.01, 0.05, 0.1%).

Extraction from the plasma samples was performed with methylene chloride.

Resumption of residue after evaporation was performed with the mobile phase.

Determination of carbamazepine and ketoconazole in biological samples of plasma was performed by liquid chromatography methods. Plasma separation methods and dosage have been developed and validated in the laboratories of the Department of Analytical Chemistry and Biopharmacy & Pharmacol Res S.A .

Results

The representation of the surfactant effects on yield of extraction, measured by the ratio between the chromatographic peak area in the presence of the tween and the peak area obtained in its absence, depending on the surfactant concentration in plasma and the analyte concentration appear in the graphs below.

Fluctuations in the area of small concentrations can not be considered artifacts or random fluctuations, first because they are found in the average curve. In addition, this type of behavior around the critical micellar concentrations (critical micellar concentration is not constant but is in function of thermodynamic parameters of the solutions studied, a very complicated function considering the complex composition of human plasma) is actually a rediscovery of the results of the first phase. In the researches on the transfer kinetics of ketoconazole from tablets in aqueous medium with addition of tween, the speed addiction and the disposal degree from the pharmaceutical form by the concentration of tween followed roughly the same structure. Other results of the first phase have also showed that the sulfur suspension stability depended on the concentration of surfactant by a complex manner, at the concentrations from the field of the critical micellar concentration of the tween occurring "critical" behaviors and at higher concentrations the stability increasing with the addition of surfactant.

CONCLUSIONS

   1. Relative representation (the ratio between the yield obtained in the presence of Tween in various concentrations and that obtained in the absence of Tween) allows one to compare the effects of surfactant at different concentrations of the analyte.
   2. At concentrations higher than the cmc, closer to the usual ones (in which surfactants are used to increase the speed and degree of dissolution, to increase the stability of disperse systems or to increase the absorption) is obtained as is expected by the similarity with other transfer phenomena mentioned, an increasing of the extraction yield with the increasing of concentration.
   3. In the vicinity of the critical micellar concentration (defined rather as concentration a range because of its dependence on a lot of kinetic and thermodynamic parameters of solutions, in our case 0.01% - 0.10%) occurred in all cases fluctuations. The reversal of the effect was rather the rule than the exception. At the same concentration of surfactant, but at different analyte concentrations, a minimum or a local maximum appeared (fig. 1, fig. 2). In the case of carbamazepine after averaging three values of analyte concentration, such reversals disappears, and in the case of ketoconazole they are preserved even in the media. Given that the results of experiments conducted in the first phase of the project as well as preliminary research related to subsequent phases, related to the dissolution kinetics, showed the same reversal of the effects in the area of low surfactant concentrations (Tween 80) the most relevant conclusion is that the reversals are real determined the association of surfactant molecules and not random fluctuations, although these reversals are not always found in the average results.
   4. Both fluctuations and the effects are greater at low concentrations of analyte (in our case 1 ppm).
   5. Effects on the kinetics of extraction liquid / liquid could not be highlighted because in bioanalytics these extractions are done in conditions of intense agitation and centrifugation. These effects occurred pregnant in the case of the dissolution of ketoconazole tablets, respectively nimesulid including concurrent extraction processes, interfacial transfer and diffusion. Slopes of the curves of dissolution increased with the increasing of the surfactant concentration, especially for pH values where the solubility of the analyte is small.
   6. The difference between carbamazepine and ketoconazole arise from their different structures and solubilities. The effect is greater on ketoconazole than on carbamazepine because carbamazepine is poorly soluble in water than ketoconazole.
   7. As a final conclusion, the surfactans effect on extraction efficiency is more than significant. The growth may reach up to 200% depending on analyte solubility and concentration of surfactant. So the accuracy of bioanalytical results may be significantly affected by the presence of the surfactants into the environment. It is expected that the results obtained with artificial surfactants are reflected in subsequent research, which will be performed with physiological surfactants.

Phase III/2010

Objectives of the phase

   1. The study of the influence of lecithin and bile salts on extraction efficiency and the distribution of a drug between plasma or physiological serum and an organic solvent.
   2. The study of the structure of some colloidal systems from mixing plasma with physiological surfactants and plasmatic lipids and of the distribution of a drug between the phases of the resulted dispersed systems.
   3. The creation of colloidal systems with a role of @experimental model@ to study the behaviour of drugs in physiological colloidal systems.

Phase activities

A.31: The study of dependence of the efficiency and kinetics extraction of the plasma depending on the nature and the concentration of the bile salts, the nature and concentration of the product of the digestion of lipids, particularly fatty acids and monoglycerides

A.3.2: The estimation of the distribution of active substances from emulsified fats and salts micelles of the bile acids

A.3.3: The design of colloidal systems to simulate plasma composition in terms of different diets associated to the clinical studies.

A.3.4: Wide-scale dissemination of the results, connection to national and international research networks, web page completion, participation in the organized training courses

RESULTS

A.31: The study of efficiency dependence and kinetics extraction of the plasma depending on the nature and concentration of bile salts, the nature and concentration of the product of the digestion of lipids, particularly fatty acids and monoglycerides

1. Extraction of ketoconazole (K) from physiological serum in CH2Cl2 in the presence of bile salts, lecithin and bile salts + lecithin. .

The representation (in fact the area's little chromatography) of the extracted amount of ketoconazole is given in Figure 1. 

Discussions

1. In the case of lecithin, the extraction efficiency decreases with increasing the concentration of lecithin. The effect is found at all concentrations of ketoconazole and then even is linear.

2. In the case of bile salts the effect is again of decreasing the extraction yield (the amount remaining and the aqueous layer is higher). The effect could even be considered as linearly dependent on the concentration of bile salts.

3. The effect of the bile salts and lecithin mixture is also lowering the yield of extraction, but less markedly than in the case when we have only lecithin. The increase does not seem to be linear with the increase of the bile salts plus lecithin concentration but rather to increase at very small concentrations and then having a small stationary. 

2. The extraction of ketoconazole (K) from plasma in CH2Cl2 in the presence of bile salts, lecithin and bile salts + lecithin. .

Conclusions:

The effect of lecithin is much higher than that of bile salts.

The bile salts added to lecithin, increase further its effect, the top of the parable being at all concentrations of ketoconazole. 

3. Estimation of partition coefficient of ketoconazole methylene chloride / plasma in the presence of bile salts, lecithin and bile salts + lecithin.

Partition coefficient changed significantly in the presence of lecithin, amplified by the presence and the effect of bile salts. The surfactants concentration effect curve is very different from concentrations of 1 mg / l and 10 mg / l ketoconazole

A.3.2: Estimated distribution of active substances between emulsified fats and micelles salts of bile acids

We have developed model systems of emulsions with lipid content closed to the natural lipids used in food, compared with synthetic oil phase emulsions and were established the optimum parameters for preparation and stability of these systems.

It was incorporated a model active ingredient in emulsion and its distribution was studied between emulsion phase and water, according to presence or absence of bile salts.

The reagents used in this study were: sodium dodecyl sulfate (SDS), polysorbate 20 (Tween 20), isopropyl myristate (IPM). The active ingredient used as a model was ketoprofen.

Following the calculation and the graphical representation for three of the samples prepared in the laboratory, for which the concentration of the surfactant ranged from 3% to 7%, it can be observed that the emulsion droplet size decreases with the increasing of the surfactant concentration.

The study of the stability of the drug ketoprofen in emulsion

To see how the surfactants influence the distribution of ketoprofen between aqueous and oily phase, samples were made with one nonionic surfactant (Tween 20) and one anionic (SDS).