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The aims of this study were to evaluate the feasibility of a nortriptyline (NT) formulation for transdermal administration and to assess the usefulness of an estimated kinetic parameter (kout) using the in vitro infinite dose technique to predict in vivo plasma levels when used in combination with pharmacokinetic parameters. To do so, a simple one-compartment model was used to describe the transport of a permeant across a membrane (skin). This model provides relatively simple expressions for the amount of permeant in the skin, the cumulative amount of permeant that crosses the skin, and the flux of permeant, for both the infinite and the finite dose regimens. Transdermal administration of the formulated NT gel to rats resulted in plasma levels of approximately 150 ng/mL between 8 and 30 h post-administration. These levels were higher than the minimum concentration of 40 ng/mL recommended for smoking cessation therapy and slightly higher than the upper limit of the therapeutic range for the treatment of depression in humans. The one-compartment model used to describe transport across the skin was connected to a two-compartment pharmacokinetic model used to predict NT plasma concentrations in rats using the kout determined in vitro and the values of other pharmacokinetic parameters obtained in vivo. The predicted concentrations were close to the observed plasma levels and the time profiles were similar for both types of data. These results show the usefulness of the kout parameter determined in vitro to predict plasma concentrations of drugs administered percutaneously.

[EN] Pistacia lentiscus L. is a sclerophyllous shrub capable of growing under harsh climatic conditions especially in the Mediterranean Basin. Different products can be obtained from this plant, such as essential oil, mastic gum or even fixed oil. The last is well known for its flavor which is mainly exploited in the food industry. Additionally, it has been traditionally used in the treatment of skin diseases, but, at the moment, any suitable formulation for skin delivery has been formulated and its biological effects was not deeply confirmed. Given that, in the present study, the lentisk oil has been formulated in liposomes at different concentrations (10, 20, 30 mg/ml) and their physicochemical, technological and main biological properties have been evaluated. Vesicles were prepared by using natural soy lecithin and a green and organic solvent free method, thus obtaining spherical, small (similar to 118 nm), homogeneously dispersed (0.27) and highly negatively charged (similar to -62 mV) vesicles. The used amount of oil loaded in liposomes (10, 20, 30 mg/ml) modulated the penetration ability of vesicles in the skin, favoring the deposition of the payload in the deeper strata. The loading in the vesicles potentiated the ability of oil to counteract the damaging effects caused by hydrogen peroxide in keratinocytes and fibroblasts and facilitate their migration in a cell monolayer lesion. Overall findings suggested that the incorporation of lentisk oil in liposomes made from soy lecithin can be an alternative and natural approach to exploit it in pharmaceutical ad cosmetical applications and manufacturing natural products suitable for the treatment of skin lesions., The authors thank MEDLENTISK project "Partnership for the exchange of best practices on lentisk tree fixed oil, an emblematic non-timber forest product in the Mediterranean" co-funded by the Erasmus+ Programme of the European Union for providing analytical equipment and materials.

International audience; Background Population pharmacokinetic evaluations have been widely used in neonatal pharmacokinetic studies, while machine learning has become a popular approach to solving complex problems in the current era of big data. Objective The aim of this proof-of-concept study was to evaluate whether combining population pharmacokinetic and machine learning approaches could provide a more accurate prediction of the clearance of renally eliminated drugs in individual neonates. Methods Six drugs that are primarily eliminated by the kidneys were selected (vancomycin, latamoxef, cefepime, azlocillin, ceftazidime, and amoxicillin) as 'proof of concept' compounds. Individual estimates of clearance obtained from population pharmacokinetic models were used as reference clearances, and diverse machine learning methods and nested cross-validation were adopted and evaluated against these reference clearances. The predictive performance of these combined methods was compared with the performance of two other predictive methods: a covariate-based maturation model and a postmenstrual age and body weight scaling model. Relative error was used to evaluate the different methods. Results The extra tree regressor was selected as the best-fit machine learning method. Using the combined method, more than 95% of predictions for all six drugs had a relative error of < 50% and the mean relative error was reduced by an average of 44.3% and 71.3% compared with the other two predictive methods. Conclusion A combined population pharmacokinetic and machine learning approach provided improved predictions of individual clearances of renally cleared drugs in neonates. For a new patient treated in clinical practice, individual clearance can be predicted a priori using our model code combined with demographic data. Bo-Hao Tang and Zheng Guan contributed equally to this work.

In the present work, red grape seed and stalk extracts were incorporated in vesicular systems designed for topical application. The phytocomplexes were obtained by maceration of biomasses in ethanol and subsequent lyophilisation. Seed extract was rich in catechin, epicatechin, epicatechin gallate, while gallic acid, epigallocatechin gallate, quercetin, quercetin 3-glucoside and malvidin-3-glucoside were detected in higher amounts in the stalk extract. Both extracts were incorporated in liposomes, hyalurosomes and transfersomes. In addition, hyalo-transfersomes were developed for the first time in this work, by combining the main modifiers of hyalurosomes and transfersomes (i.e., sodium hyaluronate and tween 80, respectively). The mean diameter of the vesicles was ∼120 nm, and the zeta potential was strongly negative (∼67 mV), irrespective of the vesicle composition or the extract used. The antioxidant activity of the extracts, measured by the DPPH (2,2-Diphenyl-1-picrylhydrazyl) test, was ∼78%, and increased (∼86%) when loaded in the vesicles. Extract loaded vesicles were biocompatible with keratinocytes and fibroblasts, the main skin cells, and were able to protect the cells from oxidative damage and promote cell proliferation and migration at the wound site.

The aim of the work was to extract, characterize, and formulate Thymus capitatus (Tymbra capitata) essential oil in phospholipid vesicles: liposomes, glycerosomes and Penetration Enhancer-containing Vesicles (PEVs). The steam-distilled essential oil was mainly composed of carvacrol. The oil was mixed with lecithin and water to produce liposomes, or different ratios of water/glycerol or water/propylene glycol (PG) to produce glycerosomes and PG-PEVs, respectively. Cryo-TEM showed the formation of unilamellar, spherical vesicles, and light scattering disclosed that their size increased in the presence of glycerol or PG, which improved long-term stability. The formulations were highly biocompatible, and capable of counteracting oxidative stress and favouring wound repair in keratinocytes, thanks to enhanced uptake. The antibacterial activity of the oil was demonstrated against cariogenic Streptococcus mutans, Lactobacillus acidophilus, and commensal Streptococcus sanguinis. The combination of antioxidant and antibacterial activities of Thymus essential oil formulations may be useful for the treatment of oral cavity diseases.

This work aimed at developing a mouthwash based on liposomes loading Citrus limon var. pompia essential oil or citral to treat oropharyngeal diseases. Vesicles were prepared by dispersing phosphatidylcholine and pompia essential oil or citral at increasing amounts (12, 25 and 50 mg/mL) in water. Transparent vesicle dispersions were obtained by direct sonication avoiding the use of organic solvents. Cryogenic transmission electron microscopy (cryo-TEM) confirmed the formation of unilamellar, spherical and regularly shaped vesicles. Essential oil and citral loaded liposomes were small in size (~110 and ~100 nm, respectively) and negatively charged. Liposomes, especially those loading citral, were highly stable as their physico-chemical properties did not change during storage. The formulations were highly biocompatible against keratinocytes, were able to counteract the damages induced in cells by using hydrogen peroxide, and able to increase the rate of skin repair. In addition, liposomes loading citral at higher concentrations inhibited the proliferation of cariogenic bacterium.

Pomace seed extract loaded vesicles were prepared as promising technological and green solution to exploit agri-food wastes and by-products, and develop high value-added products for human health. An antioxidant extract rich in bioactive compounds (epicatechins, catechin, gallic acid, quercetin and procynidins) was obtained from the seeds isolated from the pomace of Cannonau red grape cultivar. The extract was incorporated into phospholipid vesicles ad hoc formulated for intestinal delivery, by combining them, for the first time, whit a maltodextrin (Glucidex). Glucidex-transfersomes, glucidex-hyalurosomes and glucidex-hyalutransferomes were prepared, characterized and tested. Glucidex-liposomes were used as reference. All vesicles were small in size (~ 150 nm), homogeneously dispersed and negatively charged. Glucidex-transfersomes and especially glucidex-hyalutransfersomes disclosed an unexpected resistance to acidic pH and high ionic strength, as they maintained their physico-chemical properties (size and size distribution) after dilution at pH 1.2 simulating the harsh gastric conditions. Vesicles were highly biocompatible and able to counteract the oxidative damages induced in Caco-2 cells by using hydrogen peroxide. Moreover, they promoted the formation of Lactobacillus reuteri biofilm acting as prebiotic formulation. Overall results suggest the potential of glucidex-hyalutransfersomes as food supplements for the treatment of intestinal disorders.

This work aimed at developing a mouthwash based on liposomes loading Citrus limon var. pompia essential oil or citral to treat oropharyngeal diseases. Vesicles were prepared by dispersing phosphatidylcholine and pompia essential oil or citral at increasing amounts (12, 25 and 50 mg/mL) in water. Transparent vesicle dispersions were obtained by direct sonication avoiding the use of organic solvents. Cryogenic transmission electron microscopy (cryo-TEM) confirmed the formation of unilamellar, spherical and regularly shaped vesicles. Essential oil and citral loaded liposomes were small in size (~110 and ~100 nm, respectively) and negatively charged. Liposomes, especially those loading citral, were highly stable as their physico-chemical properties did not change during storage. The formulations were highly biocompatible against keratinocytes, were able to counteract the damages induced in cells by using hydrogen peroxide, and able to increase the rate of skin repair. In addition, liposomes loading citral at higher concentrations inhibited the proliferation of cariogenic bacterium.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) widely used as a component of High Active Antiretroviral Therapy (HAART) since it is inexpensive, readily absorbed after oral administration and non-teratogenic. In the present work, the mechanism of a previously described pharmacokinetic interaction between NVP and the antidepressant drug nortriptyline (NT) was studied using rat hepatic microsomes. The obtained results showed a competitive inhibition of the NVP metabolism by NT. The three main NVP metabolites (2-OH-NVP, 3-OH-NVP and 12-OH-NVP) where competitively inhibited with similar inhibitory constant values (Ki = 4.01, 3.97 and 4.40 μM, respectively). Time-dependent inhibition of the NVP metabolism was also detected, with a 2.5-fold reduction in the IC50 values of NT for 2-, 3-, and 12-OH-NVP formation when NT was preincubated with the microsomal suspension in the presence of an NADPH-generating system. A concentration-dependent inhibition of the formation of NVP metabolites by the main NT metabolite (10-OH-NT) was also observed, however, the inhibitory potency of 10-OH-NT was much lower than that of the parent drug. The apparent hepatic intrinsic clearance of NVP determined in these in vitro experiments was used to predict the in vivo clearance of NVP using the “well-stirred” and the “parallel-tube” models, resulting in values close to those previously observed in vivo clearance. Finally, a good prediction of the increase in the plasma concentrations of NVP when co-administered with NT was obtained employing the inhibitory constant of NT determined in vitro and the estimated plasma concentration of NT entering the liver.

The therapy of inflammatory bowel diseases is still rather inefficient, and about 80% of patients require surgery at some stage. Improving the treatments by more efficient medication is, therefore, an urgent medical need. The objective of this project was to demonstrate targeted delivery of Cyclosporine-A (CYA) to the inflamed areas of the intestinal mucosa after oral administration, enabling improved alleviation of the symptoms and, at the same time, reduced systemic drug absorption and associated adverse effects. As had already been demonstrated in previous studies, nano- to micrometer-sized drug particles will accumulate at inflamed mucosal areas, providing a platform for such purposes. CYA as incorporated in poly-(lactic-co-glycolic-Acid) (PLGA) nano- and mirocarriers, respectively, each homogeneous in size and providing controlled drug release over 24h at intestinal pH-value. For comparative reasons, a commercial formulation (Sandimmun Neoral®) was included in the study. In an acute model of DSS-induced inflammation in Balb/c mice, up to three doses were administered for each formulation: 50mg/kg, 25mg/kg and 12.5mg/kg. Drug-free particles were included as control. The following parameters were evaluated: body weight, colon length, colon weight/length ratio, cytokine expression and histological analysis. Plasma levels of CYA were analysed to compare systemic bioavailability. While disease parameters, such as, e.g. colon length, always improved with an optimum dose of 25mg/kg, the commercial and the microparticulate formulations led to measurable plasma levels and adverse effects in terms of body weight loss at the highest dose. In contrast, when administering the same doses as nanoparticles, plasma concentrations remained always below the detection limit, and the body weight of the animals remained unchanged. In conclusion, this study corroborates the potential of nanocarriers enabling an improved topical delivery of CYA to the inflamed gut mucosa after oral administration yielding the same improvement of disease parameters at only half the dose in comparison to microparticles and a commercial oral formulation, respectively, and at the same time minimizing systemic exposure and associated adverse effect.

The subcutaneous administration route is widely used to administer different types of drugs given its high bioavailability and rapid onset of action. However, the sensation of pain at the injection site might reduce patient adherence. Apart from a direct effect of the drug itself, several factors can influence the sensation of pain: needle features, injection site, volume injected, injection speed, osmolality, viscosity and pH of formulation, as well as the kind of excipients employed, including buffers and preservatives. Short and thin needles, conveniently lubricated and with sharp tips, are generally used to minimize pain, although the anatomic injection site (abdomen versus thigh) also affects the sensation of pain. Large subcutaneous injection volumes are associated with pain. In this sense, the maximum volume generally accepted is around 1.5 ml, although volumes of up to 3 ml are well tolerated when injected in the abdomen. Injected volumes of up to 0.5–0.8 ml are not expected to increase substantially the pain produced by the needle insertion. Ideally, injectable products should be formulated as isotonic solutions (osmolality of about 300 mOsm/kg) and no more than 600 mOs/kg have to be used in order to prevent pain. A pH close to the physiological one is recommended to minimize pain, irritation, and tissue damage. Buffers are frequently added to parenteral formulations to optimize solubility and stability by adjusting the pH; however, their strength should be kept as low as possible to avoid pain upon injection. The data available recommend the concentration of phosphate buffer be limited to 10 mM and that the concentration of citrate buffer should be lower than 7.3 mM to avoid an increased sensation of pain. In the case of preservatives, which are required in multiple-dose preparations, m-cresol seems to be more painful than benzyl alcohol and phenol. Funding: Sandoz SA.

Objectives In the absence of consensus, the present meta-analysis was performed to determine an optimal dosing regimen of vancomycin for neonates. Methods A ‘meta-model’ with 4894 concentrations from 1631 neonates was built using NONMEM, and Monte Carlo simulations were performed to design an optimal intermittent infusion, aiming to reach a target AUC0–24 of 400 mg·h/L at steady-state in at least 80% of neonates. Results A two-compartment model best fitted the data. Current weight, postmenstrual age (PMA) and serum creatinine were the significant covariates for CL. After model validation, simulations showed that a loading dose (25 mg/kg) and a maintenance dose (15 mg/kg q12h if 89% of the treated patients. Conclusions The results of a population meta-analysis of vancomycin data have been used to develop a new dosing regimen for neonatal use and to assist in the design of the model-based, multinational European trial, NeoVanc.

A new class of biocompatible and scalable phospholipid vesicles was developed, aiming at improving the efficacy of baicalin on the skin. Phosphatidylcholine and baicalin (a natural polyphenol) were hydrated in two steps with a mixture of ethanol, glycerol, and propylene glycol at different ratios, and a low amount of water (4%). Hence, water was almost completely replaced by the co-solvents, which were never used before as predominant dispersing medium of phospholipid vesicles. The vesicles appeared three-dimensionally structured, forming a network that conferred a high viscosity to the dispersions. The vesicles were unilamellar, small in size (∼100 nm), and stable during 12 months of storage. They disclosed optimal performances in the transdermal delivery of baicalin, and high biocompatibility with skin cells (i.e., keratinocytes and fibroblasts). Furthermore, the vesicles promoted the efficacy of baicalin in protecting skin cells against oxidative stress in vitro and injured skin in vivo.

New three-dimensionally-structured hybrid phospholipid vesicles, able to load clotrimazole in a high amount (10 mg/mL), were obtained for the first time in this work by significantly reducing the amount of water (&le, 10%), which was replaced with a mixture of glycerol and ethanol (&asymp, 90%). A pre-formulation study was carried out to evaluate the effect of both the composition of the hydrating medium and the concentration of the phospholipid on the physico-chemical properties of hybrid vesicles. Four different three-dimensionally-structured hybrid vesicles were selected as ideal systems for the topical application of clotrimazole. An extensive physico-chemical characterization performed using transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), 31P-NMR, and small-angle X-ray scattering (SAXS) displayed the formation of small, multi-, and unilamellar vesicles very close to each other, and was capable of forming a three-dimensional network, which stabilized the dispersion. Additionally, the dilution of the dispersion with water reduced the interactions between vesicles, leading to the formation of single unilamellar vesicles. The evaluation of the in vitro percutaneous delivery of clotrimazole showed an improved drug deposition in the skin strata provided by the three-dimensionally-structured vesicles with respect to the commercial cream (Canesten&reg, ) used as a reference. Hybrid vesicles were highly biocompatible and showed a significant antifungal activity in vitro, greater than the commercial cream Canesten&reg, The antimycotic efficacy of formulations was confirmed by the reduced proliferation of the yeast cells at the site of infection in vivo. In light of these results, clotrimazole-loaded, three-dimensionally-structured hybrid vesicles appear to be one of the most innovative and promising formulations for the treatment of candidiasis infections.

Aim The adequate dosing of topiramate in neonates undergoing therapeutic hypothermia has not been established. The aim of this study was to design a dosing schedule capable of providing topiramate serum concentrations within the accepted therapeutic range. Methods Neonates (n = 52) with hypoxic ischaemic encephalopathy and subjected to therapeutic hypothermia were dosed with topiramate, 5 mg/kg on day one and 3 mg/kg on days two to five, to decrease seizure events. A total of 451 topiramate serum concentrations obtained in the patients were used to develop a population pharmacokinetic model using a non-linear mixed-effects modelling approach. Results A one-compartment model with first-order absorption and two different clearance terms, one for the cooling period and another for the post-warming period, were used to describe the concentration-time topiramate data. The probability of no-seizure events could not be related to topiramate concentrations, which was attributed to excessively low topiramate concentrations. A modified dosage schedule was designed with the aim of obtaining more than 90% of patients with topiramate concentrations within the therapeutic range after the first dose. Conclusion The dosage schedule of topiramate in these patients should be modified with the aim of decreasing the frequency of seizure events.

The present study aimed at developing a new vesicular formulation capable of promoting the protective effect of ascorbic acid and tocopherol against intestinal oxidative stress damage, and their efficacy in intestinal wound healing upon oral administration. A pH-dependent copolymer (Eudragit&reg, L100), a water-soluble prebiotic fibre (Nutriose&reg, FM06), a phospholipid mixture (Lipoid S75), and two natural antioxidants (ascorbic acid and tocopherol) were combined to fabricate eudragit-nutriosomes by a simple, solvent-free procedure. The vesicles were spherical and oligolamellar, with some multicompartment structures in Eudragit-nutriosomes, small in size (~100 nm), with highly negative zeta potential. The effect of Eudragit&reg, and Nutriose&reg, on the stability on storage and in simulated gastrointestinal fluids were confirmed by the Turbiscan&reg, technology and in vitro studies, respectively. Eudragit-nutriosomes exhibited a protective effect against H2O2-induced oxidative stress, and a proliferative effect in Caco-2 cells, as they provided the closure of the scratched area after 96 h of incubation.

Magnolia spp. extracts are known for their use in traditional Korean, Chinese, and Japanese medicine in the treatment of gastrointestinal disorders, anxiety, and allergies. Among their main components with pharmacological activity, the most relevant are magnolol and honokiol, which also show antitumoral activity. The objectives of this work were to study some physicochemical properties of both substances and their stability under different conditions of temperature, pH, and oxidation. Additionally, liposomes of honokiol (the least stable compound) were formulated and characterized. Both compounds showed pH-dependent solubility, with different solubility–pH profiles. Magnolol showed a lower solubility than honokiol at acidic pH values, but a higher solubility at alkaline pH values. The partition coefficients were similar and relatively high for both compounds (log Po/w ≈ 4.5), indicating their lipophilic nature. Honokiol was less stable than magnolol, mainly at neutral and basic pH values. To improve the poor stability of honokiol, it was suitably loaded in liposomes. The obtained liposomes were small in size (175 nm), homogeneous (polydispersity index = 0.17), highly negatively charged (−11 mV), and able to incorporate high amounts of honokiol (entrapment efficiency = 93.4%). The encapsulation of honokiol in liposomes increased its stability only at alkaline pH values.

Citrus species extracts are well known sources of bio-functional compounds with health-promoting effects. In particular, essential oils are known for their antibacterial activity due to the high content of terpenes. In this work, the steam-distilled essential oil from the leaves of Citrus limon var. pompia was loaded in phospholipid vesicles. The physico-chemical characteristics of the essential oil loaded vesicles were compared with those of vesicles that were loaded with citral, which is one of the most abundant terpenes of Citrus essential oils. The biocompatibility of the vesicles was assessed in vitro in human keratinocytes. Furthermore, the antimicrobial activity of the vesicles was tested while using different bacterial strains and a yeast: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans, respectively. The vesicles were small in size (~140 nm), slightly polydispersed (PI ~0.31), highly negatively charged (~ &minus, 73 mV), and able to incorporate high amounts of essential oil or citral (E%~86%). Pompia essential oil and citral exhibited antimicrobial activity against all of the assayed microorganisms, with P. aeruginosa being the least sensitive. Citral was slightly more effective than pompia essential oil against E. coli, S. aureus, and C. albicans. The incorporation of citral in vesicles improved its antifungal activity against C. albicans.

Aiming at improving the protective effects of baicalin on the skin, new highly-biocompatible penetration enhancer containing vesicles (PEVs) were developed by modifying the base formulation of transfersomes with sorbitol, thus obtaining sorbitol-PEVs. An extensive evaluation of the physico-chemical features of both transfersomes and sorbitol-PEVs was carried out. Transfersomes were mainly close-packed, multi-compartment vesicles, while sorbitol-PEVs appeared mostly as single, spherical, unilamellar vesicles. All the vesicles were small in size (∼128 nm) and negatively charged (∼−67 mV), without significant differences between the formulations. The in vitro delivery of baicalin to intact skin showed an improved ability of sorbitol-PEVs to favour the deposition of the flavonoid into the whole skin. In addition, the vesicular formulations protected keratinocytes and fibroblasts from oxidative stress and UV radiation, and promoted cell proliferation and migration, which favoured the closure of skin wound. Cell uptake was promoted as well, especially when sorbitol-PEVs were used.

Transfersomes were prepared by using different polysorbates (i.e., Tween 20, 40, 60 and 80) and loaded with tocopherol acetate, a naturally-occurring phenolic compound with antioxidant activity. The vesicles showed unilamellar morphology, small size (∼85 nm), low polydispersity index (≤0.27), and high entrapment efficiency, which increased as a function of the length of the Tween fatty acid chain (from 72% to 90%). The long-term stability of the formulations was evaluated by means of the Turbiscan™ technology, which indicated their good stability, irrespective of the Tween used. The vesicles efficiently delivered tocopherol to the skin, and showed biocompatibility in vitro in keratinocytes and fibroblasts. Regardless of the Tween used, the transfersomes were able to protect skin cells from the oxidative damage induced by hydrogen peroxide. Additionally, transfersomes promoted cell proliferation and migration, which resulted in an acceleration of skin wound closure. These results demonstrated that tocopherol-loaded transfersomes bear potential as topical delivery system with antioxidant activity and wound healing properties.

Nutriosomes, new phospholipid nanovesicles specifically designed for intestinal protection were developed by simultaneously loading a water-soluble dextrin (Nutriose® FM06) and a natural antioxidant (curcumin). Nutriosomes were easily fabricated in a one-step, organic solvent-free procedure. The stability and delivery performances of the vesicles were improved by adding hydroxypropyl methylcellulose. All the vesicles were small in size (mean diameter ∼168 nm), negatively charged (zeta potential ∼-38 mV, irrespective of their composition), and self-assembled predominantly in unilamellar vesicles stabilized by the presence of Nutriose®, which was located in both the inter-lamellar and inter-vesicle media, as confirmed by cryo-TEM and SAXS investigation. The dextrin acted also as a cryo-protector, avoiding vesicle collapse during the lyophilization process, and as a protector against high ionic strength and pH changes encountered in the gastrointestinal environment. Thanks to the antioxidant properties of curcumin, nutriosomes provided an optimal protective effect against hydrogen peroxide-induced oxidative stress in Caco-2 cells. Moreover, these innovative vesicles showed promising efficacy in vivo, as they improved the bioavailability and the biodistribution of both curcumin and dextrin upon oral administration, which acted synergically in reducing colonic damage chemically induced in rats. © The Royal Society of Chemistry 2018., SAXS experiments were performed at the BL11-NCD beamline of the ALBA Synchrotron facility with the collaboration of ALBA staff. The beam-time at ALBA was kindly provided within the approved proposal no. 2013110789. The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under the grant agreement no. 312284.

One of the major sources of flavonoids for humans are citrus fruits, hesperidin being the predominant flavonoid. Hesperetin (HSP), the aglycon of hesperidin, has been reported to provide health benefits such as antioxidant, anti-inflammatory and anticarcinogenic effects. However, the effect of HSP on skin pigmentation is not clear. Some authors have found that HSP induces melanogenesis in murine B16-F10 melanoma cells, which, if extrapolated to in vivo conditions, might protect skin against photodamage. Since the effect of HSP on normal melanocytes could be different to that observed on melanoma cells, the described effect of HSP on murine melanoma cells has been compared to the effect obtained using normal human melanocytes. HSP concentrations of 25 and 50 µM induced melanin synthesis and tyrosinase activity in human melanocytes in a concentration-dependent manner. Compared to control melanocytes, 25 µM HSP increased melanin production and tyrosinase activity 1.4-fold (p

One of the most frequent comorbidities of HIV infection is depression, with a lifetime prevalence of 22 to 45%. Therefore, it was decided to study a potential pharmacokinetic interaction between the nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) and the tricyclic antidepressant nortriptyline (NT). NVP and NT were administered to rats either orally, intraduodenally, or intravenously, and the changes in plasma levels and pharmacokinetic parameters were analyzed. Experiments with rat and human hepatic microsomes were carried out to evaluate the inhibitory effects of NT on NVP metabolism. NVP plasma concentrations were significantly higher when this drug was coadministered with NT. The maximum plasma concentrations of NVP were increased 2 to 5 times and the total plasma clearance was decreased 7-fold in the presence of NT. However, statistically significant differences in the pharmacokinetic parameters of NT in the absence and presence of NVP were not found.In vitrostudies with rat and human hepatic microsomes confirmed the inhibition of NVP hepatic metabolism by NT in a concentration-dependent way, with the inhibition being more intense in the case of rat microsomes. In conclusion, a pharmacokinetic interaction between NVP and NT was detected. This interaction was a consequence of the inhibition of hepatic metabolism of NVP by NT.In vivohuman studies are required to evaluate the effects of this interaction on the pharmacokinetics of NVP before it can be taken into account for patients receiving NVP.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in the treatment of human immunodeficiency virus type 1 (HIV-1) and is the first-choice NNRTI during pregnancy. NVP shows a sex dimorphic profile in humans with sex differences in bioavailability, biotransformation and toxicity. In this study, sex differences in NVP metabolism and inhibition of NVP metabolism by the antidepressant nortriptyline (NT) were evaluated using rats as experimental animals. NVP was administered orally to male and female rats and sex differences in plasma levels and pharmacokinetic parameters were analysed. NVP plasma levels were higher in female compared with male rats, and pharmacokinetic parameters such as maximum plasma concentration (Cmax), time to Cmax (Tmax), half-life (t1/2) and area under the plasma concentration–time curve from the time of dosing to the last measurable concentration (AUClast) showed ca. 4-, 5-, 7- and 22-fold higher values in female rats. In vitro experiments carried out with hepatic microsomes confirmed slower NVP metabolism in female rats, with a maximum velocity (Vmax) 2-fold lower than in male hepatic microsomes. The major metabolite in both sexes was 12-hydroxynevirapine (12-OH-NVP), with the Vmax for this metabolite being 15-fold lower in female compared with male rat hepatic microsomes. Inhibition of NVP metabolism by NT was similar in both sexes, with statistically non-significant differences in 50% inhibitory concentration (IC50) values. In summary, NVP is metabolised more slowly in female compared with male rats, but the inhibitory effect of NT is similar in both sexes.

[EN] Resveratrol and gallic acid, a lipophilic and a hydrophilic phenol, were co-loaded in innovative, biocompatible nanovesicles conceived for ensuring the protection of the skin from oxidative-and inflammatory-related affections. The basic vesicles, liposomes and glycerosomes, were produced by a simple, one-step method involving the dispersion of phospholipid and phenols in water or water/glycerol blend, respectively. Liposomes and glycerosomes were modified by the addition of poloxamer, a stabilizer and viscosity enhancer, thus obtaining viscous or semisolid dispersions of structured vesicles. The vesicles were spherical, unilamellar and small in size (similar to 70 nm in diameter). The superior ability of the poloxamer-structured vesicles to promote the accumulation of both phenols in the skin was demonstrated, as well as their low toxicity and great ability to protect fibroblasts from chemically-induced oxidative damage. The in vivo administration of the vesicular phenols on TPA (phorbol ester)-exposed skin led to a significant reduction of oedema and leukocyte infiltration. (C) 2017 Elsevier Inc. All rights reserved., This work was partially supported by grants BIO2014-52872-R (Ministerio de Economia y Competitividad, Spain), which included FEDER funds, and 2014-SGR-938 (Generalitat de Catalunya, Spain).

Glycerosomes were formulated using 1,2-dimyristoyl-sn-glycero-3-phosphatidycholine (DMPC), diclofenac sodium salt and 10, 20 or 30% glycerol in the water phase, while corresponding liposomes were prepared with the same amount of DMPC and diclofenac, without glycerol. The aim of the present work was to evaluate the effect of the used phospholipid on vesicle features and ability to favour diclofenac skin deposition by comparing these results with those found in previous works performed using hydrogenated soy phosphatidylcholine (P90H) and dipalmitoylphosphatidylcholine (DPPC). Liposomes and glycerosomes were multilamellar, liposomes being smaller (72±6nm). Interactions among glycerol, phospholipids and drug led to the formation of a non-rigid bilayer structure and a variation of the main transition temperature, which shifted to lower temperature. The addition of glycerol led to the formation of more viscous systems (from ∼2.5mPa/s for basic liposomes to ∼5mPa/s for glycerosomes), which improved spread ability of the formulations on the skin.Results obtained in vitro were promising using glycerosomes, irrespective of the amount of glycerol used: the amount of drug, which accumulated into and permeated through the different skin strata, was high and comparable with that obtained using P90H, suggesting that glycerosomes may represent an efficient carrier for both local effect or systemic absorption.

Vancomycin is one of the most evaluated antibiotics in neonates using modeling and simulation approaches. However no clear consensus on optimal dosing has been achieved. The objective of the present study was to perform an external evaluation of published models, in order to test their predictive performances in an independent dataset and to identify the possible study-related factors influencing the transferability of pharmacokinetic models to different clinical settings. Published neonatal vancomycin pharmacokinetic models were screened from the literature. The predictive performance of 6 models was evaluated using an independent dataset (112 concentrations from 78 neonates). The evaluation procedures used simulation-based diagnostics (visual predictive check [VPC] and normalized prediction distribution errors [NPDE]). Differences in predictive performances of models for vancomycin pharmacokinetics in neonates were found. The mean of NPDE for 6 evaluated models were 1.35, -0.22, -0.36, 0.24, 0.66, 0.48, respectively. These differences were explained, at least partly, by taking into account the method used to measure serum creatinine concentrations. The adult conversion factor of 1.3 (enzymatic to Jaffe) was tested with an improvement in the VPC and NPDE, but it still need to be evaluated and validated in neonates. Differences were also identified between analytical methods for vancomycin. The importance of analytical techniques for serum creatinine concentrations and vancomycin as a predictor of vancomycin concentrations in neonates has been confirmed. Dosage individualisation of vancomycin in neonates should consider not only patients’ characteristics and clinical conditions, but also the methods used to measure serum creatinine and vancomycin.

Resveratrol and gallic acid were co-loaded in phospholipid vesicles aiming at protecting the skin from external injuries, such as oxidative stress and microbial infections. Liposomes were prepared using biocompatible phospholipids dispersed in water. To improve vesicle stability and applicability, the phospholipids and the phenols were dispersed in water/propylene glycol or water/glycerol, thus obtaining PEVs and glycerosomes, respectively. The vesicles were characterized by size, morphology, physical stability, and their therapeutic efficacy was investigated in vitro. The vesicles were spherical, unilamellar and small in size: liposomes and glycerosomes were around 70nm in diameter, while PEVs were larger (∼170nm). The presence of propylene glycol or glycerol increased the viscosity of the vesicle systems, positively affecting their stability. The ability of the vesicles to promote the accumulation of the phenols (especially gallic acid) in the skin was demonstrated, as well as their low toxicity and great ability to protect keratinocytes and fibroblasts from oxidative damage. Additionally, an improvement of the antimicrobial activity of the phenols was shown against different skin pathogens. The co-loading of resveratrol and gallic acid in modified phospholipid vesicles represents an innovative, bifunctional tool for preventing and treating skin affections.

This work aimed at finding an innovative vesicle-type formulation able to improve the bioavailability of curcumin upon oral administration. To this purpose, phospholipid, Eudragit® S100 and hyaluronan sodium salt were combined to obtain eudragit-hyaluronan immobilized vesicles using an easy and environmentally-friendly method. For the first time, the two polymers were combined in a system intended for oral delivery, to enhance curcumin stability when facing the harsh environment of the gastrointestinal tract. Four different formulations were prepared, keeping constant the amount of the phospholipid and varying the eudragit-hyaluronan ratio. The freeze-drying of the samples, performed to increase their stability, led to a reduction of vesicle size and a good homogeneity of the systems, after simple rehydration with water. X-ray diffraction study demonstrated that after the freeze-drying process, curcumin remained successfully incorporated within the vesicles. All the vesicles displayed similar features: size ranging from 220 to 287nm, spherical or oval shape, multilamellar or large unilamellar morphology with a peculiar multicompartment organization involving 1-4 smaller vesicles inside. In vitro studies demonstrated the ability of the combined polymers to protect the vesicles from the harsh conditions of the gastro-intestinal tract (i.e., ionic strength and pH variation), which was confirmed in vivo by the greater deposition of curcumin in the intestinal region, as compared to the free drug in dispersion. This enhanced accumulation of curcumin provided by the eudragit-hyaluronan immobilized vesicles, together with an increase in Caco-2 cell viability exposed to hydrogen peroxide, indicated that vesicles can ensure a local protection against oxidative stress and an increase in its intestinal absorption.

Allantoin is traditionally employed in the treatment of skin ulcers and hypertrophic scars. In the present work, to improve its local deposition in the skin and deeper tissues, allantoin was incorporated in conventional liposomes and in new argan oil enriched liposomes. In both cases, obtained vesicles were unilamellar, as confirmed by cryo-TEM observation, but the addition of argan oil allowed a slight increase of the mean diameter (∼130nm versus ∼85nm). The formulations, especially those containing argan oil, favoured the allantoin accumulation in the skin, in particular in the dermis (∼8.7μg/cm(2)), and its permeation through the skin (∼33μg/cm(2)). The performances of vesicles as skin delivery systems were compared with those obtained by water dispersion of allantoin and the commercial gel, Sameplast(®). Moreover, in this work, for the first time, the elastic and viscous moduli of the skin were measured, underlining the different hydrating/moisturizing effects of the formulations. The application of ARG liposomes seems to provide a softening and relaxing effect on the skin, thus facilitating the drug accumulation and passage into and trough it.

A simple, rapid, and sensitive HPLC method based on 9H-fluoren-9-ylmethyl chloroformate derivatization for the quantification of sertraline in rat plasma has been developed, requiring a plasma sample of only 0.1 mL, which was deproteinized and derivatized for 5 min in two single steps. The obtained derivative was stable at room temperature and was determined by HPLC using a fluorescence detector. The analytical column was a C(18) column and the mobile phase was acetonitrile and water (80:20, v/v). Calibration curves were linear in the range of 10-500 ng/mL. The limit of detection was approximately 3 ng/mL, and the lower limit of quantification was established at 10 ng/mL. The bias of the method was lower than 10%, and the within day as well as between day, relative standard deviations were lower than 12%. This analytical method was successfully applied to characterize sertraline pharmacokinetics in rats following intravenous (t(1/2) = 213 ± 48 min, Cl = 43.1 ± 8.7 mL/min, V(d) = 11560 ± 1861 mL) and oral (C(max) = 156 ± 76 ng/mL, t(max) = 63.8 ± 16.3 min) administration of 2 and 5 mg, respectively.

An in vivo preclinical study has been made of the oral absorption of morphine (CAS 57-27-2) from a new sustained release formulation (morphine-Eudragit® L complex, MEC), which had shown good sustained release properties in in vitro dissolution studies. The absorption of morphine from capsules filled with morphine hydrochloride trihydrate (MHT) or MEC was compared in fasted and fed dogs. Mean plasma morphine concentrations obtained after administration of MHT and MEC to fasted dogs were similar, and no statistically significant differences were found in the pharmacokinetic parameters of morphine (C max , T max and area under the plasma morphine concentration versus time curve from time zero to the last time with a detectable concentration of morphine). When MHT and MEC were administered to fed animals, mean plasma morphine concentrations were again similar for both formulations, without statistically significant differences in the pharmacokinetic parameters of morphine. These results contrast with those obtained in vitro , and indicate the limited usefulness of in vitro assays for this kind of sustained release formulations in which pH and ionic strength are important factors for drug release from the polymeric structure. The plasma morphine concentrations obtained in fed dogs were generally lower than in fasted dogs, though they were detectable for a longer time, until 10 h after dosing, in contrast to up to 6 h in fasted dogs. It is postulated that the apparently prolonged absorption of morphine in fed dogs may be due to the enterohepatic recycling of the drug (excreted in bile as glucuronide, hydrolysed back to the parent compound in the intestine, and then reabsorbed) as a consequence of gallbladder emptying induced by food.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Vancomycin is a glycopeptide antibiotic commonly used to treat resistant gram-positive infections in neonates. While adult dosing guidelines are generally well established, a lack of consensus for optimal dosing regimens in neonates remains. • The large variance in pharmacokinetic values in premature neonates compared with full-term infants is a major barrier to the development of optimal dosing regimens. Pharmacokinetic values have been reported for vancomycin in neonates. However, the studies have included small groups with differing, clinical conditions, serum sampling times and pharmacokinetic models. There are many proposed neonatal dosing guidelines for vancomycin, but few, if any, have been prospectively evaluated. In addition, the complexity of the dose and interval selection has led to errors in use. WHAT THIS STUDY ADDS • The purpose of this investigation was to determine population pharmacokinetic parameters of vancomycin in neonatal patients with a wide range of gestational age and birth weight and to study the effect of the co-administration of amoxicillin-clavulanic acid and spironolactone on the pharmacokinetics of vancomycin in this cohort of patients. The aim was to find covariates with a relevant influence on the pharmacokinetic parameters of this drug, and to use this information for the design of an initial dosing schedule of vancomycin in neonates. Additionally, the obtained population pharmacokinetic parameters can be used to perform modifications of the initial dosing schedule in neonates with serum concentrations outside of the therapeutic range, by means of a Bayesian approach. AIM To determine the population pharmacokinetic parameters of vancomycin in neonatal patients with a wide range of gestational age and birth weight, and subsequently to design an initial dosing schedule for vancomycin in neonates. METHODS Using nonlinear mixed-effects modelling (NONMEM VI), the pharmacokinetics of vancomycin were investigated in 70 neonates with postmenstrual age and body weight ranging 25.1–48.1 weeks and 0.7–3.7 kg, respectively. A one-compartment linear disposition model with zero order input and first-order elimination was used to describe the data. Nine demographic characteristics and 21 co-administered drugs were evaluated as covariates of clearance (CL) and distribution volume (Vd) of vancomycin. RESULTS Weight-normalized clearance of vancomycin was influenced by postmenstrual age (PMA) and co-administration of amoxicillin-clavulanic acid. Weight-normalized volume of distribution was influenced by co-administration of spironolactone. CL and Vd of the typical individual in this study population (PMA = 34.6 weeks, weight = 1.7 kg) were estimated to be 0.066 l h−1 kg−1 (95% CI 0.059, 0.073 l h−1 kg−1) and 0.572 l kg−1 (95% CI 0.505, 0.639 l kg−1), respectively. This model was used to predict a priori serum vancomycin concentrations in a validation group (n= 41), which were compared with observed concentrations to determine the predictive performance of the model. The 95% confidence interval of mean prediction error included zero for both peak and trough vancomycin concentrations. CONCLUSIONS Postmenstrual age, co-administration of amoxicillin-clavulanic acid and spironolactone have a significant effect on the weight-normalized CL and Vd. An initial dosage guideline for vancomycin is proposed for preterm and full-term neonates, whereas the population pharmacokinetic model can be used for dosage individualization of vancomycin.

A simple, sensitive and specific high-performance liquid chromatography method has been developed for the determination of nortriptyline (NT) in plasma samples. The assay involved derivatization with 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl) and isocratic reversed-phase (C 18 ) chromatography with fluorescence detection. The developed method required only 100 μl of plasma sample, deproteinized and derivatized in one step. Calibration curves were lineal over the concentration range of 5–5000 ng/ml. The derivatization reaction was performed at room temperature in 20 min and the obtained NT derivative was stable for at least 48 h at room temperature. The within-day and between-day relative standard deviation was below 8%. The limit of detection (LOD) was 2 ng/ml, and the lower limit of quantification (LLOQ) was established at 10 ng/ml. The method was applied on plasma collected from rats, at different time intervals, after intravenous administration of 0.5 mg of NT.

Microparticles of chitosan (CHT) containing alendronate sodium (AL) were prepared in four drug:polymer ratios (1:1, 1:2, 1:4, 1:6) using the spray drying technique. The efficiency of the method was evaluated by determining production yield (about 70 %) and microencapsulation efficiency, which was almost 100 % in the case of all four of the formulations studied. Particles had a mean size of between 3.6 and 4.6 microm, and a near-spherical shape. The formulations with the highest content of AL (drug:polymer ratio 1:1 and 1:2) showed an asymmetrical distribution of particles, which were larger in size, and had a higher proportion of irregular particles than the other formulations. FT-IR analysis revealed an ionic interaction between AL and CHT. Differential scanning calorimetry and thermogravimetric analysis confirmed the microencapsulation of AL and the increased thermal stability of encapsulated AL. The dissolution profiles of AL from CHT microspheres, at pH values of 1.2 and 6.8, showed a delayed release of AL from microspheres, and the dissolution rate was dependent on the pH and the drug:polymer ratio. It can be concluded that spray drying is a suitable technique for preparing AL-loaded CHT microspheres, and that the drug:polymer ratio can be used to control the rate of AL release from microspheres.

Between 22 and 45% of HIV-positive subjects are likely to report symptoms of depression. Considering this background, a potential pharmacokinetic interaction between the nonnucleoside reverse transcriptase inhibitor efavirenz (EFV) and two antidepressants, sertraline (SRT) and nortriptyline (NT), was studied. Rats were administered EFV alone or together with the antidepressants, and changes in the plasma levels and pharmacokinetic parameters of EFV were analyzed. Additional in vitro experiments with rat and human hepatic microsomes were carried out to evaluate the inhibitory effect of SRT and NT on EFV metabolism by determining the formation rate of the major EFV metabolite (8-OH-EFV). In vivo studies showed similar increases in the plasma levels of EFV when it was coadministered with SRT or NT. However, the studies using rat hepatic microsomes showed a more potent inhibitory effect of NT than of SRT on the metabolism of EFV, with values for the 50% inhibition constant (IC 50 ) and inhibitory constant ( K i ) for NT about 9-fold lower than those for SRT. An equation was deduced that explains the similar in vivo effects of SRT and NT in spite of the different in vitro performance data. Using human hepatic microsomes, the strongest inhibitory effect was observed with SRT. In summary, pharmacokinetic interactions between EFV, SRT, and NT, associated with the inhibition of hepatic metabolism of EFV, have been detected in rats. Both antidepressants also inhibit EFV metabolism in human hepatic microsomes, but additional in vivo studies in humans are required to evaluate the clinical implication of this interaction.

The aim of the study was to characterize the platelet count (PLT) dynamics in peritoneal carcinomatosis patients treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal oxaliplatin (HIO). Data from patients treated with CRS alone (N = 18) or CRS and HIO (N = 62) were used to estimate the baseline platelet count (PLT0), rate constants for platelet maturation (k tr ) and platelet random destruction (k s ), feedback on progenitor cell proliferation (γ), and the drug-specific model parameters (α, β). Plasma oxaliplatin concentrations, C p , reduced the proliferation rate of progenitor cells (k prol) according to a power function α × C p (β) . The surgery effect on k prol and k s was explored. The typical values (between subject variability) of the PLT0, k tr , k s , γ, α, and β were estimated to be 237 × 10(9) cells/L (32.9%), 7.09 × 10(-3) h(-1) (47.1%), 8.86 × 10(-3) h(-1) (80.0%), 0.621, 0.88 L/mg (56.9%), and 2.63. Surgery induced a maximal 2.09-fold increase in k prol that was attenuated with a half-life of 8.42 days. Splenectomy decreased k s by 47.5%. Age, sex, body surface area, sex, total proteins, and HIO carrier solution did not impact the model parameters. The model developed suggests that, following CRS and HIO, thrombocytopenia and thrombocytosis were reversible and short-lasting; the severity of the thrombocytopenia and thrombocytosis was inversely correlated, with splenectomized patients having thrombocytopenia of lower severity and thrombocytosis of higher severity; and the HIO dose and treatment duration determine the severity and duration of the thrombocytopenia. Higher HIO dose or longer treatment duration could be used without substantially increasing the risk of major hematological toxicity.

In this study, we characterize the population pharmacokinetics of kahalalide F (KF), a novel marine anticancer drug, after intravenous (i.v.) administration in advanced cancer patients. Data from 240 patients included in three Phase I and three Phase II trials receiving i.v. weekly and every 3 weeks infusions of KF, at doses ranging 266–6650 µg/m2, were analyzed using NONMEM™ VII. The effect of demographics and/or pathophysiologically relevant factors on KF pharmacokinetic parameters was evaluated. Model evaluation was conducted using nonparametric bootstrap and visual predictive check (in both internal and external datasets). An open two-compartment model with linear distribution and elimination from central compartment was suitable to describe the data. Volume of distribution at steady state and its between-subject variability (CV%) was estimated to be 6.56 L (28 %). Plasma clearance was estimated to be 6.25 L/h (43 %). Within the range of covariates evaluated, age, weight, body surface area, gender, ECOG performance status, presence of liver metastases, creatinine clearance, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total bilirubin, total protein and serum albumin did not contribute to explain KF pharmacokinetic variability to a significant extent. The developed model was deemed appropriate to describe the time course of KF plasma concentrations and its variability in advanced cancer patients. The integration of pharmacokinetic data from six clinical studies demonstrated KF linear elimination from plasma, dose-proportional exposure and time-independent pharmacokinetics. Based on analyzed data, no clinically relevant covariates were identified as predictors of KF pharmacokinetics.

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37°C in the presence of DNase I and sera from the three species. Both high pH and heparin were able to release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI–DNA and DOTAP–DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds.