Prototyping Trastuzumab Docetaxel Immunoliposomes with a New FCM-Based Method to Quantify Optimal Antibody Density on Nanoparticles.

Developing targeted nanoparticles is a rising strategy to improve drug delivery in oncology. Antibodies are the most commonly used targeting agents. However, determination of their optimal number at the surface remains a challenging issue, mainly due to the difficulties in measuring precisely surface coating levels when prototyping nanoparticles. We developed an original quantitative assay to measure the exact number of coated antibodies per nanoparticle. Using flow cytometry optimized for submicron particle analysis and beads covered with known amounts of human IgG-kappa mimicking various amounts of antibodies, this new method was tested as part of the prototyping of docetaxel liposomes coated with trastuzumab against Her2+ breast cancer. This quantification method allowed to discriminate various batches of immunoliposomes depending on their trastuzumab density on nanoparticle surface (i.e., 330 (Immunoliposome-1), 480 (Immunoliposome-2) and 690 (Immunoliposome-3), p = 0.004, One-way ANOVA). Here we showed that optimal number of grafted antibodies on nanoparticles should be finely tuned and highest density of targeting agent is not necessarily associated with highest efficacy. Overall, this new method should help to better prototype third generation nanoparticles.

"No pain, No gain" still true with immunotherapy: When the finger shows the moon, look at the moon!

There is a rising evidence that the proverbial statement "No pain, No gain" first coined at the light of pioneering clinical experiences with canonical chemotherapy still holds true in the era of modern treatments of cancer. This close relationship between the occurrence of specific drug-related toxicity and treatment outcome has been confirmed since then with a large variety of treatments, ranging from cytotoxics, hormonotherapy, targeted therapy and much interestingly even with the latest immune checkpoint inhibitors. In the current context of precision medicine, and along with the constant quest for identifying predictive biomarkers, close monitoring of treatment-related toxicities could therefore be convenient to help predicting therapeutic response, but presents several caveats. The purpose of this review is to briefly describe these relationships across the different treatments, to comment on possible underlying mechanisms and to comment on possible strategies aiming at exploiting this relationship while keeping the maximal safety ensured in patients with cancer. In particular, this review will investigate on how drug exposure along with germinal and somatic genetic issues does impact on the "No Pain, No Gain" aphorism, and why the temptation to use treatment-related toxicities as a cheap and convenient way to predict clinical outcome or to adapt dosing should be resisted. We do advocate instead for developing comprehensive genomic support along with extensive biomathematical modeling to better customize dosing and shift towards a new "No Pain, Maximal Gain" paradigm.

Population pharmacokinetics model of THC used by pulmonary route in occasional cannabis smokers.

Cannabis is the most widely used illegal drug in the world. Delta-9-tetrahydrocannabinol (THC) is the main source of the pharmacological effect. Some studies have been carried out and showed significant variability in the described models as the values of the estimated pharmacokinetic parameters. The objective of this study was to develop a population pharmacokinetic model for THC in occasional cannabis smokers. Twelve male volunteers (age: 20-28years, body weight: 62.5-91.0kg), tobacco (3-8 cigarette per day) and cannabis occasional smokers were recruited from the local community. After ad libitum smoking cannabis cigarette according a standardized procedure, 16 blood samples up to 72h were collected. Population pharmacokinetic analysis was performed using a non-linear mixed effects model, with NONMEM software. Demographic and biological data were investigated as covariates. A three-compartment model with first-order elimination fitted the data. The model was parameterized in terms of micro constants and central volume of distribution (V1). Normal ALT concentration (6.0 to 45.0IU/l) demonstrated a statistically significant correlation with k10. The mean values (%Relative Standard Error (RSE)) for k10, k12, k21, k23, k32 and V1 were 0.408h-1 (48.8%), 4.070h-1 (21.4%), 0.022h-1 (27.0%), 1.070h-1 (14.3%), 1.060h-1 (16.7%) and 19.10L (39.7%), respectively. We have developed a population pharmacokinetic model able to describe the quantitative relationship between administration of inhaled doses of THC and the observed plasma concentrations after smoking cannabis. In addition, a linear relationship between ALT concentration and value of k10 has been described and request further investigation.

Acceleration of clear cell renal cell carcinoma growth in mice following bevacizumab/Avastin treatment: the role of CXCL cytokines.

The anti-VEGF targeted antibody bevacizumab (BVZ) has been approved for treating renal cell carcinomas (RCCs). Although BVZ increases the progression-free survival of patients with metastatic RCC, the effect on overall survival is poor. To gain insight into the limited efficacy of BVZ on overall survival, we analyzed patient samples of RCC for angiogenic factors that may participate in escape from anti-VEGF therapy. Our study shows that the level of vascular endothelial growth factor (VEGF) in tumors was increased compared with normal tissue. The level of interleukin-8/CXCL8, a pro-angiogenic member of the CXCL family of cytokines, was also increased in tumors. These observations gave us a good reason to analyze the combined effects of BVZ and anti-CXCL8 antibodies on tumor growth. Surprisingly, we report that BVZ accelerates the growth of RCC in nude mice with in vivo selection of tumor cells with an increased growth capacity. Downregulation of receptor tyrosine phosphatase-κ, a phosphatase implicated in EGF receptor regulation, may partly explain this phenomenon. Modification of the vascular network and development of lymphatic vessels through VEGF-C production and compensatory production of pro-angiogenic CXCL cytokines were also observed. The apparent normalization of the vascular network prompted us to associate BVZ with the chemotherapeutic agent paclitaxel. While efficient in vitro, paclitaxel did not reverse the anti-VEGF effects in vivo. Anti-CXCL8-targeting antibodies were promising as they decreased intra-tumor VEGF production; decreased the pro-angiogenic CXCL/anti-angiogenic CXCL ratio and did not induce lymphangiogenesis. These observations hold clinical implication as they highlight putative markers implicated in escape from BVZ treatment. They also recommend proceeding with caution in the use of anti-VEGF therapy alone for treatment of RCC.

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method for routine clinical monitoring of tacrolimus with the Waters Masstrak™ immmunosuppressant kit.

Advances in mass spectrometry instruments have led to increased utilization of high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and it would be necessary to standardize blood quantification of immunosuppressant drugs. The aim of the study was to validate and assess the robustness of an LC-MS/MS method for quantification of tacrolimus in whole blood using the Waters Masstrak™ Immunosuppressant Kit. After protein precipitation from whole blood samples, chromatographic separation was performed in 2 min. Detection was performed with a Waters Tandem Quadrupole MS Quattro Premier XE, operated in multiple-reaction monitoring in positive electrospray ionization mode. This method was validated and compared to Enzyme Multiplied Immunoassay Technique (EMIT) method in accordance with actual guidelines. The limit of quantification was 1.0 ng/mL and the calibration curve was linear to 27.6 ng/mL. Between-day and within-day trueness and precision were < 15% at three concentrations spanning the linear range. The EMIT assay showed an average positive bias of 28.3% compared with the LC-MS/MS. Internal and external quality control were always accepted and demonstrated the robustness of this method. In conclusion, we validated a rapid, simple and robust quantification of tacrolimus in blood samples with the Waters Masstrak™ Immunosuppressant Kit.

Development and validation of HPLC method for the determination of Cyclosporin A and its impurities in Neoral capsules and its generic versions.

Cyclosporin A (CyA) is a cornerstone immunosuppressant for the prophylaxis against allograft rejection after organ transplantation. The most widely prescribed CyA formulation is Neoral soft gelatine capsules (Novartis Pharmaceuticals, Basel, Switzerland). After Novartis patent expiration, several generic formulations have been developed. In this paper, a simple and reliable HPLC method was developed and validated for the evaluation of four CyA degradation products (ID-005-95, CyH, IsoCyH and IsoCyA) and two related compounds (CyB and CyG) aimed for the quality control of Neoral capsules and its generic formulations. In a second step, the validated method was then compared to the USP assay method for capsules, where some of the mentioned impurities were not adequately resolved from the CyA peak. Isocratic elution at a flow rate of 1.0mLmin(-1) was employed on a Lichrospher RP-18 (4mmx250mm; 5microm) analytical column maintained at 75 degrees C with a tetrahydrofuran:phosphoric acid (0.05M) (44:56, v/v) as mobile phase. The chromatograms were recorded using a Hewlett Packard 1100 chromatographic system. The UV detection wavelength was performed at 220nm and 10microL of sample was injected. The developed method was validated in terms of selectivity, linearity, precision, accuracy, limit of detection and limit of quantitation. The validate method was successfully applied to commercial capsules, Neoral and generic versions. Therefore, the proposed method is suitable for the simultaneous determination of CyA as well as its major impurities.

Ethnic differences in the distribution of CYP3A5 gene polymorphisms.

The genetic polymorphism affecting the CYP3A5 enzyme is responsible for interindividual and interethnic variability in the metabolism of CYP3A5 substrates. The full extent of the CYP3A5 genetic polymorphism was analysed in French Caucasian, Gabonese and Tunisian populations using a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) strategy. In the three populations, eight, 17 and ten single nucleotide polymorphisms (SNPs), respectively, were identified, among which nine correspond to rare new mutations. Also identified were 16 alleles including eight new allelic variants. Significant differences were observed in the distribution of these alleles. Particularly, the frequency of the CYP3A5*3C null allele in French Caucasians (81.3%) and in Tunisians (80.0%) is higher than in the Gabonese population (12.5%) (p < 0.001). Considering the CYP3A5 genotypes of the tested individuals, only 10.4% of French Caucasians and 30.0% of Tunisians were identified as CYP3A5 expressors. In contrast, 90.0% of Gabonese subjects appear to express the CYP3A5 protein.

Seventy-two-hour stability of Taxol in 5% dextrose or 0.9% sodium chloride in Viaflo, Freeflex, Ecoflac and Macoflex N non-PVC bags.

BACKGROUND AND OBJECTIVES: Taxol (paclitaxel), is an antimicrotubule agent widely prescribed for the treatment of many tumoral diseases. Taxol must be used in non-polyvinyl chloride bags, diluted to concentrations of 0.3-1.2 mg/mL in 5% dextrose or in 0.9% sodium chloride. Under these conditions, Taxol is chemically and physically stable for 27 h at 25 degrees C. The aim of the study was to evaluate the 72-h stability of Taxol under common clinical use conditions. METHODS: Taxol was diluted with 5% dextrose and 0.9% sodium chloride to final concentrations of 0.3 and 1.2 mg/mL in four polyolefin bags (Viaflo, Freeflex, Ecoflac and Macoflex N). Taxol-stability, was assessed by turbidimetry and by high-performance liquid chromatography using solutions stored in the dark, over 72 h at +4 degrees C. RESULTS: No haze, turbidity, or precipitate was observed. Paclitaxel concentration remained above 95% of the initial value whatever the solvent or container used. CONCLUSION: Paclitaxel at 0.3 and 1.2 mg/mL in 5% dextrose and in 0.9% sodium chloride is stable in Viaflo, Freeflex, Ecoflac and Macoflex N non-PVC bags for 72 h in the dark at +4 degrees C. The longer stability should make the use of Taxol in clinical practice easier.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-alpha and 2 PPRE sites.

BACKGROUND AND AIMS: Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. METHODS: The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. RESULTS: We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. CONCLUSIONS: We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

A simple and rapid high-performance liquid chromatographic (HPLC) method for 5-fluorouracil (5-FU) assay in plasma and possible detection of patients with impaired dihydropyrimidine dehydrogenase (DPD) activity.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) gene polymorphism may lead to severe toxicity with 5-fluorouracil (5-FU), a major anticancer drug extensively used in clinical oncology. Drug monitoring combined with early detection of patients at risk would enable timely dose adaptation so as to maintain drug concentrations within a therapeutic window. However, the best method to identify such patients remains to be determined. OBJECTIVE: The aim of this study was to develop a rapid and simple high-performance liquid chromatographic (HPLC) method for estimating uracil/dihydrouracil (U/UH2) ratio in plasma, as an index of DPD status, and for assaying 5-FU as part of drug level monitoring. METHOD: Assay of 5-FU, and U/UH2 detection were performed on a HPLC system equipped with UV detector. Analytes were separated at room temperature using a 5 microm particles, 25 cm RP-18 X-Terra column. The mobile-phase consisted of a KH(2)PO(4) salt solution (0.05 m) + 0.1% triethylamine (TEA) pumped at 0.4 mL/min. Detection of 5-FU and 5-bromouracil were performed at 254 nm; U and UH2 elution was monitored at 210 nm. RESULTS: The method was sensitive and specific for assaying 5-FU within the 5-500 ng/mL concentration range, which covers exposure levels currently met in clinical practice. The method was simple, and relatively cheap, and rapid, with an analytical run time of about 30 min. Data from a patient with 5-FU toxicity suggest that the method was capable of identifying DPD metabolic phenotype in cancer patients, based on measurement of plasma U/UH2 ratio. CONCLUSION: The method described should be suitable both for detecting patients at high risk of 5-FU toxicity, and for drug level monitoring during chemotherapy.

Experience of a combination including indinavir 400 mg plus ritonavir 200 mg twice daily in HIV-infected patients: pharmacokinetic data.

Low doses of ritonavir, a strong inhibitor of cytochrome P450 3A4, enhances the pharmacokinetic profile of indinavir with increased serum levels. We assessed the indinavir-ritonavir 400/200 twice daily combination in 17 HIV-infected patients focusing on the pharmacokinetic data and the tolerance of this regimen. IDV trough and peak concentrations were measured by high-performance liquid chromatography. Median indinavir trough and peak concentrations were 553 ng/ml and 3626 ng/ml, respectively. A good tolerance was observed except for three patients who experienced a major toxicity. Only one dose adjustment was related to indinavir toxicity. Considering the fact that Cmax is mainly responsible of the adverse effects, particularly renal stones, the indinavir-ritonavir 400/200 mg twice daily regimen offers a well-tolerated combination with an increased Cmin but a lower Cmax compared with both the standard tid regimen and higher dose of IDV-RTV regimens.

Physician recognition of active drug use in HIV-infected patients is lower than validity of patient's self-reported drug use.

A French survey of 325 HIV-infected subjects with a history of injecting drugs allowed us to study the recognition of patients' injection drug use (IDU) by physicians providing HIV-infection care, and to analyze the correlation between patient demographics and incorrect IDU identification. Kappa for concordance of physician's reports of their patient's IDU with patient's declaration was 0.37; concordance was lower among socially vulnerable patients. This contrasted with a nested study of validity of patient's self-report of opioid use: Kappa for patient's declaration of opioid use within the past two days against a biological assay was 0.61, and concordance was higher among socially vulnerable patients. Concordance of physicians' ratings and patients' reports of IDU was not more than fair, even though physicians were knowledgeable about their patient's IDU history. This concordance varied with social status in a way that did not correspond with variations in self-reported opioid use validity, suggesting that identification of active IDU might be partly based on incorrect interpretation of subjective cues.

Pharmacokinetics of sabeluzole and dextromethorphan oxidation capacity in patients with severe hepatic dysfunction and healthy volunteers.

AIMS: The primary objective of this study was to determine how the pharmacokinetics of sabeluzole, an investigational drug with specific effects on memory and learning abilities, are affected by chronic liver disease. Since sabeluzole is metabolised by CYP2D6, a secondary objective was to study the correlation between CYP2D6 activity (as assessed by the dextromethorphan dextrorphan metabolic ratio) and hepatic dysfunction. METHODS: The single-dose pharmacokinetics of sabeluzole (10 mg) was compared in 10 healthy Caucasian subjects and 10 patients with severe hepatic dysfunction. The urinary dextromethorphan/dextrorphan (DMP/DRP) metabolic ratio was determined after intake of 20 mg dextromethorphan (NODEX capsules). RESULTS: The terminal half-life of sabeluzole was significantly prolonged in subjects with severe hepatic dysfunction vs healthy subjects (respectively 39.3 +/- 11.5 h; 17.5 +/- 10.2 h (mean +/- s.d.)). The areas under the curve (AUC) were significantly higher in subjects with severe hepatic dysfunction than in healthy volunteers (681 +/- 200 ng ml(-1) h vs 331 +/- 282 ng ml(-1) h). There was a significant correlation between the AUC(0,infinity) and the DMP/DRP metabolic ratio in healthy volunteers and subjects with severe hepatic dysfunction. AUC was greater and elimination of sabeluzole slower in poor metabolizers compared with extensive metabolizers. CONCLUSIONS: These results suggest that a) sabeluzole dose should be reduced in patients with severe hepatic dysfunction and b) the AUC of sabeluzole is linked to individual CYP2D6 activity.

Plasma, urine and skin pharmacokinetics of cefepime in burns patients.

We studied the pharmacokinetics of cefepime (2 g bd) in six burns patients. Blood, urine and skin samples were collected to measure cefepime concentrations. A two-compartment model was fitted to the data. At day 1, t(1/2beta) was 2.45 +/- 0.56 h, V(ss) 0.36 +/- 0.1 L/kg, total clearance 152 +/- 25.2 mL/min, and AUC 217 +/- 34 mg*h/L. There was no statistical difference between day 1 and day 3 for any of the pharmacokinetic parameters. We demonstrated good penetration of cefepime in skin. These results show that it is not necessary to change the standard dosage of cefepime in burns patients.

Relationship between efficacy, tolerance, and plasma drug concentration of ritonavir in children with advanced HIV infection.

The relationship between ritonavir plasma concentration, efficacy, and tolerance was evaluated in 31 children with advanced HIV infection who were receiving a triple therapy with ritonavir as protease inhibitor. Median CD4+ lymphocyte count and median viral load before the initiation of ritonavir-containing combination therapy were 1320 cells/mL and 5 log10 copies/mL, respectively. Ritonavir was given at a dose ranging from 300 to 450 mg/m2 twice daily. The median follow-up of triple therapy was 19 months. Response was defined as a drop of viremia of more than 1 log. Plasma drug levels were determined twice during the observation period: after at least 4 weeks and after 3 months of combined treatment. Samples were collected before (residual) and 2 hours (T2) after drug intake. Cholesterol, triglycerides, alanine transaminase, aspartate transaminase, and gamma-glutamyl transpeptidase were assessed at the same time. The median values of ritonavir residual and T2 levels were 1.64 mg/L and 5.9 mg/L at observation 1 and 3.35 mg/L and 6.29 mg/L at observation 2, respectively. According to virologic response, median residual concentrations of ritonavir were 3.17, 2.52, and 1.04 mg/L for the complete, the partial, and the no-response groups. The authors observed a wide intersubject variability of ritonavir concentrations with an increase in residual levels between the two observation periods. Residual levels were correlated with virologic response whereas there was no direct association between T2 levels and long-term response. Patients with complete or partial response displayed statistically significantly higher residual concentrations than the no-response group. No correlation could be demonstrated between elevated plasma drug concentrations and abnormal cholesterol or triglycerides values. These results emphasize the importance of a sustained high ritonavir concentration to achieve optimal treatment efficacy. Furthermore, these results prove the clinical benefit of therapeutic drug monitoring and could potentially improve patient evaluation in terms of treatment efficacy, compliance, and viral resistance.

Cerebrospinal fluid penetration and pharmacokinetics of vancomycin administered by continuous infusion to mechanically ventilated patients in an intensive care unit.

Cerebrospinal fluid (CSF) penetration and the pharmacokinetics of vancomycin were studied after continuous infusion (50 to 60 mg/kg of body weight/day after a loading dose of 15 mg/kg) in 13 mechanically ventilated patients hospitalized in an intensive care unit. Seven patients were treated for a sensitive bacterial meningitis and the other six patients, who had a severe concomitant neurologic disease with intracranial hypertension, were treated for various infections. Vancomycin CSF penetration was significantly higher (P < 0.05) in the meningitis group (serum/CSF ratio, 48%) than in the other group (serum/CSF ratio, 18%). Vancomycin pharmacokinetic parameters did not differ from those obtained with conventional dosing. No adverse effect was observed, in particular with regard to renal function.