Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab.

BACKGROUND: Ipilimumab, an immune checkpoint inhibitor targeting CTLA-4, prolongs survival in a subset of patients with metastatic melanoma (MM) but can induce immune-related adverse events, including enterocolitis. We hypothesized that baseline gut microbiota could predict ipilimumab anti-tumor response and/or intestinal toxicity. PATIENTS AND METHODS: Twenty-six patients with MM treated with ipilimumab were prospectively enrolled. Fecal microbiota composition was assessed using 16S rRNA gene sequencing at baseline and before each ipilimumab infusion. Patients were further clustered based on microbiota patterns. Peripheral blood lymphocytes immunophenotypes were studied in parallel. RESULTS: A distinct baseline gut microbiota composition was associated with both clinical response and colitis. Compared with patients whose baseline microbiota was driven by Bacteroides (cluster B, n = 10), patients whose baseline microbiota was enriched with Faecalibacterium genus and other Firmicutes (cluster A, n = 12) had longer progression-free survival (P = 0.0039) and overall survival (P = 0.051). Most of the baseline colitis-associated phylotypes were related to Firmicutes (e.g. relatives of Faecalibacterium prausnitzii and Gemmiger formicilis), whereas no colitis-related phylotypes were assigned to Bacteroidetes. A low proportion of peripheral blood regulatory T cells was associated with cluster A, long-term clinical benefit and colitis. Ipilimumab led to a higher inducible T-cell COStimulator induction on CD4+ T cells and to a higher increase in serum CD25 in patients who belonged to Faecalibacterium-driven cluster A. CONCLUSION: Baseline gut microbiota enriched with Faecalibacterium and other Firmicutes is associated with beneficial clinical response to ipilimumab and more frequent occurrence of ipilimumab-induced colitis.

Protein binding of indomethacin in human cerebrospinal fluid.

The binding of the non-steroidal anti-inflammatory drug indomethacin to proteins in human cerebrospinal fluid (CSF), drawn during lumbar puncture from 10 patients affected by lumbosciatica, was measured by equilibrium dialysis and spectrofluorimetry. Similar binding studies on human serum albumin solutions (0.5 and 1 g/L) were performed using the same techniques. The mean binding percentage of indomethacin determined by equilibrium dialysis was 40%. The results obtained by both techniques allowed us to conclude that the binding of indomethacin in CSF was essentially due to albumin.

Representation of affinity in the case of co-operativity in protein-ligand binding.

We have already proposed a "Global Association Function" to represent the global affinity of proteins to a drug; it was first applied in the case of independent binding sites. In this paper, we show that this same function can also be used to assess interactions between sites by varying the number of interacting sites and their co-operativity level. The resulting curves in two application cases are given together with the corresponding Scatchard plot: i) in a system with one single class of identical and interacting sites, ii) in a system with two classes of sites in which either primary or secondary are interacting; unexpectedly, in this latter case we also observed that sometimes positive co-operativity occasionally resulted in a concave-up Scatchard plot which is unusually admitted. In addition, as described in one example, our function is assumption free; this might be an advantage over usual methods, such as discrete parameter methods, because they require additional and empirical hypotheses on their related binding model.

Estimation by high-performance liquid chromatography of ketoprofen in plasma. Application to the study of its protein binding.

A method of estimation by high-performance liquid chromatography of ketoprofen in plasma is proposed. This method is rapid, simple, and very sensitive: it allows concentrations of 2 ng/ml to be determined. The internal standard is (benzoyl-4-phenyl)-2-butyric acid. The drug is extracted from a biological fluid in ether, which is then evaporated; the residue is taken up in chromatographic eluant (acetonitrile/phosphate buffer pH 7), analysed by reverse-phase chromatography and detected by spectrophotometry at 256 nm. In spite of the very strong protein binding of this drug, this method allowed the determination of its free concentration in plasma.

Association and transfer functions for analysis of drug binding to proteins.

A global association function is proposed that characterizes the apparent overall equilibrium of drug protein binding. It is defined as a function of the total drug concentration and may be directly calculated from each experimental result. The binding transfer function represents the relative rates of change in the bound and free concentrations from the total concentration. Two applications are presented: one is an investigation of the effect of temperature on binding of salicylic acid to proteins in plasma; and the other is a comparison of the phenomenon in two different biological fluids (protein binding of sodium salicylate in plasma and in synovial fluid).

Difficulties in applying the Scatchard model of ligand binding to proteins--proposal of new mathematical tools--application to salicylates.

Ill-considered use of the Scatchard model often leads to unjustified deductions. Since the main difficulty of this model is its number of parameters, new models are proposed that have only two parameters. After checking the models on simulated data, they were applied to real data on the binding of salicylates to albumin.

Drug assay in ground tissues: example of ketoprofen diffusion into tonsillar tissue.

Ketoprofen was assayed in tissues of surgical patients after mechanical grinding of the tissue in liquid nitrogen; the fine powder obtained allowed the drug to be determined by HPLC in the same way as for liquid samples. The method was applied to the study of ketoprofen diffusion into the tonsillar tissue of 15 patients after a single intramuscular injection of ketoprofen (100 mg). A correction was made for blood contamination after hemoglobin determination.

Decrease of in vitro serum protein binding of salicylate in rheumatoid arthritis.

Free and bound fractions of salicylates were separated by equilibrium dialysis and measured by spectrofluorimetry in 27 patients with rheumatoid arthritis and in 16 controls. The results showed that in patients with rheumatoid arthritis, the binding of salicylate to proteins decreased in an overproportional manner with the decrease of serum albumin concentrations. This phenomenon was linked with the severity of the inflammatory syndrome. The saturation binding capacity per unit of protein concentration was lower in the patients suffering from active forms of the condition, a finding which suggests that the changes observed are not due only to quantitative changes in the serum albumins. This study confirms the importance of determining free salicylate concentrations in the treatment of patients with inflammatory diseases.

[Passage of nonsteroidal anti-inflammatory agents across the synovial membrane]. / Le passage des anti-inflammatoires non stéroïdiens à travers la synoviale.

The therapeutic effectiveness of non-steroid anti-inflammatory (NSAI) drugs is partly determined by their passage across the synovial membrane. The synovium can be compared to a double barrier the permeability of which to NSAI drugs depends on the degree of inflammation of the joint and on the pharmacokinetic properties of the drugs (lipophilia, pka, protein-binding). A few hours after one single systemic dose, concentrations in the synovial fluid are higher than in serum. During chronic administration, concentrations of NSAI drugs with a short half-life vary less in synovial fluid than in serum. During steady state, free fractions of NSAI drugs with prolonged half-life may be similar in both compartments.

Sera with high levels of anti-smooth muscle and anti-mitochondrial antibodies frequently bind to cytoskeleton proteins.

Using ELISA methods, 54 sera from chronic active hepatitis (CAH) patients displaying high levels of anti-smooth muscle antibodies (SMA) and 18 sera from primary biliary cirrhosis (PBC) patients with high levels of anti-M2 antibodies were examined for the presence of high antibody levels against actin, tubulin, myosin, tropomyosin, troponin, vimentin and desmin. Our results showed that: (i) in CAH with high SMA activity, increased antibody levels were found in 51.9% of sera for actin, 31.5% for myosin, 35.2% for tubulin, 34.0% for tropomyosin, 11.3% for troponin, 22.6% for vimentin and 43.4% for desmin, compared with natural antibody levels in 21 normal sera; (ii) Similar high levels of these antibodies were found in the case of PBC; (iii) in most cases, sera simultaneously bound to several antigens of the panel; and (iv) approximately 26% of the CAH sera were found to be negative with the seven antigens examined while 22% were reacted with a cytoskeleton protein (CP) other than actin. These results indicate that current opinion associating SMA with anti-actin activity in CAH is confirmed for only 50% of cases and that although a good correlation between SMA and anti-CP antibodies can be obtained, there is still a significant percentage of SMA for which the putative antigen recognized needs to be determined.

Dangers and problems in calculating coefficients of a sum of exponential functions.

The use of the computer in making calculations has led increasingly to the estimation of parameters of exponential functions from point experimental measurements. This involves the use of techniques such as logarithmic transformations and criteria (especially that of the least squares), of which the use is not always justified. Radioactivity measurements raise special problems as they are subject to a statistical distribution of the Poisson type. The authors propose a method based on the statistical criterion of "maximum likelihood" which permits tests of the number of exponentials from point measurements (in practice, this method is valid for one or two exponentials), and also the calculation of parameters more satisfactorily than customary methods.

Direct high-performance liquid chromatographic resolution of the enantiomers of tiaprofenic acid using immobilized human serum albumin.

Resolution of racemic tiaprofenic acid (TA) has been performed using immobilized human serum albumin as the stationary phase. The eluent was phosphate buffer-acetonitrile-n-octanoic acid (90:10:0.015, v/v). Detection was achieved at 305 nm. The pharmacokinetics of the enantiomers were studied following oral administration into humans and after subcutaneous injection in rats. Plasma concentrations of (+)-TA were much greater than those of (-)-TA. For the rat, the pharmacokinetic parameters between (-)-TA and (+)-TA were all statistically different (p < 0.005).

[Determination of protein binding of drugs using equilibrium dialysis. Influence of volume displacement caused by osmotic pressure]. / Détermination de la fixation protéique des médicaments par dialyse à l'équilibre. Influence du déplacement de volume sous l'effet de la pression osmotique.

By the determination of the free concentration of a drug using the equilibrium dialysis method, a volume shift could be observed, inducing a dilution of the protein medium. The value of this volume shift varies with the plasmatic sample and could produce a misdetermination of the free fraction which could be important, essentially for the drugs strongly bound to proteins.

Stereoselective binding of etodolac to human serum albumin.

The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1 to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the (R)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations, S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and II, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site II.

Albumin binding sites for etodolac enantiomers.

Non-steroidal anti-inflammatory drugs (NSAIDs) are strongly bound to human serum albumin (HSA), mainly to sites I and II. The aim of this study was to characterize the binding site(s) of etodolac enantiomers under physiological conditions (580 microM HSA) using equilibrium dialysis. The protein binding of etodolac enantiomers, alone or in various ratios, was studied in order to evaluate the potential competition between them. Our results showed that (S)-etodolac was more strongly bound to HSA than (R)-etodolac. The displacement of one enantiomer by its antipode was observed only at high concentrations of the competitor, and was more pronounced for (S)-form. Displacement studies of the enantiomers by specific probes of sites I and II of albumin, dansylamide, and dansylsarcosine, respectively, showed that (R)-etodolac was slightly displaced by both these probes whereas the free concentration of (S)-etodolac increased markedly in the presence of dansylsarcosine. Moreover, the binding of ligands to sites I and II is usually affected by alkaline pH, by chloride ions, and by fatty acids. For etodolac, the presence of 0.1 and 1 M chloride ions and increasing pH (5.5-9) decreased the binding of both enantiomers. The same result was obtained with addition of octanoic acid. Conversely, the addition of oleic, palmitic, or stearic acid to the protein solution increased the binding of (R)-etodolac, but decreased that of its antipode. All these findings suggest (R)- and (S)-etodolac interact mainly with site II of HSA, and that the (R)-isomer is also bound to site I under physiological conditions.

The effect of food on the systemic availability of ketoprofen.

We have studied the pharmacokinetics of ketoprofen, a non-steroidal anti-inflammatory drug, in 12 patients after a single 100 mg oral dose both in fasting conditions and with a meal. Food significantly affected the peak plasma concentration of ketoprofen and decreased its absorption rate. However, the extent of absorption of ketoprofen, as reflected by the area under the plasma concentration time curve, appeared to be unchanged in the presence of food.

Effect of age on the disposition of sodium fluoride.

Sodium fluoride (NaF) is used in the treatment of axial osteoporosis and so is mostly given to old patients. Since its pharmacokinetics has not been studied in the elderly, the pharmacokinetics of an enteric-coated tablet containing 50 mg NaF has been investigated in 15 aged inpatients (aged 65 to 75 y) and 12 young healthy volunteers (aged 21 to 26 y). The serum AUC of fluoride was 1.7-time higher in older than in younger subjects. There was a strong inverse correlation between the AUC and either body surface area (BSA) or glomerular filtration rate (GFR), both of which were very much lower in the elderly. This concluded that if efficacy or safety are related to the bioavailability of fluoride, it may be valuable to adjust the dosage of fluoride accordingly to the GFR and BSA.

Differences in sodium salicylate protein binding in serum and synovial fluid from patients with a knee effusion.

Protein binding of sodium salicylate in synovial fluid and in serum from 23 inpatients with rheumatic diseases were studied, ex vivo, by equilibrium dialysis. Scatchard model with two classes of sites was used as a mathematical tool. At therapeutic concentrations, protein binding of sodium salicylate was significantly higher in serum than in synovial fluid. The ratio of areas under the curves for bound concentrations for synovial fluid to that for serum was 0.867. This difference was attributed to the hypoalbuminemia observed in the synovial fluid; for a given molar ratio of drug to albumin, or in other words, for the same amount of available drug per mole of albumin. The number of sites occupied was the same in the two biological media.

[Protein binding of salicylates in rheumatoid arthritis]. / Fixation protéique des salicylés au cours de la polyarthrite rhumatoïde.

Protein binding of salicylates was determined in 16 control subjects and 27 patients suffering from rheumatoid arthritis. Results obtained after separation of the free and bound fractions by dialysis to equilibrium and measured by spectrofluorometry were analyzed using a new mathematical model. In correlation with the decrease in plasma albumin concentrations, a decrease was found in the protein binding of salicylates in patients with rheumatoid arthritis. This phenomenon was less marked in therapeutic zones and was related to the degree of severity of the inflammatory syndrome. The binding capacity per albumin molecule at saturation was decreased in patients suffering from advanced forms, suggesting that the changes seen were not due solely to quantitative variations in serum albumin levels. This study confirms the value of the determination of free salicylate levels in patients suffering from inflammatory rheumatic disorders.