Identification and characterization of the enzymes responsible for the metabolism of the non-steroidal anti-inflammatory drugs, flunixin meglumine and phenylbutazone, in horses.

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug-drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450-mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma-hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5-OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5-OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug-drug interactions and identification of reasons for varying pharmacokinetics between horses.

Role of Herborn (K240E) and Milano Slow (D375H) human serum albumin variants towards binding of phenylbutazone and ibuprofen.

Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA determines the pharmacokinetics and pharmacodynamics of the drugs. There are 67 natural genetic variants of HSA were reported in literature. Studying the effect of albumin modifications on drug binding helps to treat the patients with proper medication. In the present study, we have aimed to understand the effect of two natural variants of HSA, such as Herborn (K240E) and Milano Slow (D375H) on the binding of phenylbutazone and ibuprofen. For this, we have generated K240E and D375H mutants and also double mutant (K240E/D375H) of HSA using site directed mutagenesis. The recombinant HSA and its variants were expressed in Pichia pastoris. The interaction of HSA and its variants to phenylbutazone and ibuprofen was studied using fluorescence spectroscopy. Our results showed that there is no significant effect of K240E and D375H mutations on phenylbutazone and ibuprofen binding. But the effect is significant when both the mutations were there in a single protein (K240E/D375H). Further, the CD spectroscopy data showed that there is no effect of phenylbutazone and ibuprofen binding on the conformation of protein, except in case of D375H, where there is a conformational change in the binding pocket with the ibuprofen binding.

Toward of Safer Phenylbutazone Derivatives by Exploration of Toxicity Mechanism.

A drug design for safer phenylbutazone was been explored by reactivity and docking studies involving single electron transfer mechanism, as well as toxicological predictions. Several approaches about its structural properties were performed through quantum chemistry calculations at the B3LYP level of theory, together with the 6-31+G(d,p) basis sets. Molecular orbital and ionization potential were associated to electron donation capacity. The spin densities contribution showed a preferential hydroxylation at the para-positions of phenyl ring when compared to other positions. In addition, on electron abstractions the aromatic hydroxylation has more impact than alkyl hydroxylation. Docking studies indicate that six structures 1, 7, 8 and 13⁻15 have potential for inhibiting human as well as murine COX-2, due to regions showing similar intermolecular interactions to the observed for the control compounds (indomethacin and refecoxib). Toxicity can be related to aromatic hydroxylation. In accordance to our calculations, the derivatives here proposed are potentially more active as well safer than phenylbutazone and only structures 8 and 13⁻15 were the most promising. Such results can explain the biological properties of phenylbutazone and support the design of potentially safer candidates.

Oxidative Alteration of Trp-214 and Lys-199 in Human Serum Albumin Increases Binding Affinity with Phenylbutazone: A Combined Experimental and Computational Investigation.

Human serum albumin (HSA) is a target for reactive oxygen species (ROS), and alterations of its physiological functions caused by oxidation is a current issue. In this work, the amino-acid residues Trp-214 and Lys-199, which are located at site I of HSA, were experimentally and computationally oxidized, and the effect on the binding constant with phenylbutazone was measured. HSA was submitted to two mild oxidizing reagents, taurine monochloramine (Tau-NHCl) and taurine dibromamine (Tau-NBr2). The oxidation of Trp-214 provoked spectroscopic alterations in the protein which were consistent with the formation of N'-formylkynurenine. It was found that the oxidation of HSA by Tau-NBr2, but not by Tau-NHCl, provoked a significant increase in the association constant with phenylbutazone. The alterations of Trp-214 and Lys-199 were modeled and simulated by changing these residues using the putative oxidation products. Based on the Amber score function, the interaction energy was measured, and it showed that, while native HSA presented an interaction energy of -21.3 kJ/mol, HSA with Trp-214 altered to N'-formylkynurenine resulted in an energy of -28.4 kJ/mol, and HSA with Lys-199 altered to its carbonylated form resulted in an energy of -33.9 kJ/mol. In summary, these experimental and theoretical findings show that oxidative alterations of amino-acid residues at site I of HSA affect its binding efficacy.

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi-spectroscopic and computational investigation.

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH 7.4) by multi-spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, Kb , value was found to lie between 2.69 × 103 and 9.55 × 103  M-1 at experimental temperatures (293, 298, 303, and 308 K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub-domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH0 ) and entropy change (ΔS0 ) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril-BSA interaction, and 8-anilino-1-naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3-dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction.

Intermolecular interactions determined by NOE build-up in macromolecules from hyperpolarized small molecules.

The nuclear Overhauser effect (NOE) is a primary means to characterize intermolecular interactions using modern NMR spectroscopy. Multiple experiments measured using different mixing time can be used for quantifying NOE buildup and measuring cross-relaxation rates. However, this approach using conventional multi-dimensional NMR is time consuming. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) can generate deviations from equilibrium spin polarization by orders of magnitude, thereby enhancing signals and allowing to characterize NOE build up in real-time. Since most small molecules can be hyperpolarized using D-DNP, this method is applicable to the study of intermolecular interactions between small molecules and macromolecules. This application is demonstrated using a model system for host-guest interactions including the third generation polyamidoamine dendrimer (G3 PAMAM) and the pharmaceutical phenylbutazone (PBZ). After mixing 1H hyperpolarized PBZ with PAMAM, the NOE build up is directly observed at different sites of the dendrimer in series of one-dimensional NMR spectra. Cross-relaxation rates specific to individual source and target spins are determined from the build up curves. Further, the polarization enhancement is shown to be sufficiently large to allow identification of cross-peaks not observed in a conventional 2D-NOESY spectrum. The improved signal-to-noise ratio provided by hyperpolarization allows for characterizing the intermolecular interaction in an almost instantaneous measurement, opening an application to macromolecular and biomacromolecular NMR.

Unravelling the binding mechanism and protein stability of human serum albumin while interacting with nefopam analogues: a biophysical and insilico approach.

In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in the blood. Its binding affinity and concomitant changes in its conformation, binding site and simulations were also studied. Fluorescence data revealed that the fluorescence quenching of HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs binding constants (KA) are in the order of NF3 > NF2 > NF1 with values of 1.53 ± .057 × 104, 2.16 ± .071 × 104 and 3.6 ± .102 × 105 M-1, respectively. Concurrently, thermodynamic parameters indicate that the binding process was spontaneous, and the complexes were stabilized mostly by hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with hydrophobic interactions. Circular dichroism (CD) studies revealed that there is a decrease in α-helix with an increase in ß-sheets and random coils signifying partial unfolding of the protein upon binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular docking studies showed that NF1, NF2 and NF3 bound to subdomains IIIA, IB and IIA through hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with LYS 413. Furthermore, molecular simulations unveiled that NFs binding was in support with the structural perturbation observed in CD, which is evident from the root mean square deviation and Rg fluctuations. We hope our insights will provide ample scope for engineering new drugs based on the resemblances with NFs for enhanced efficacy with HSA.

Phenylbutazone, a New Long-Acting Agent that can Improve the Peptide Pharmacokinetic Based on Serum Albumin as a Drug Carrier.

As a NPY-2 receptor agonist, PYY24-36- Leu31 is reported to suppress appetite and has a potential in obesity treatment, but its short half-life limits the clinical application. The use of chemical modification to improve interactions with human serum albumin (HSA) is an effective strategy for prolonging the half-lives of peptide analogues. So based on the characteristics that phenylbutazone has a good combination with HSA, we selected a proper linker to link with PYY24-36 -Leu31 to create long-acting and highly biologically active PYY24-36 -Leu31 conjugates, and successfully find a novel, long-acting PYY24-36 -Leu31 conjugate 8 that, when dosed every other day in diet induce obese (DIO) mice for 2 weeks, results in a significant reduction in food intake and body weight and improvement in blood parameter and hepatic steatosis.

Phenylbutazone Oxidation via Cu,Zn-SOD Peroxidase Activity: An EPR Study.

We investigated the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD)-peroxidase activity on the oxidation of the nonsteroidal anti-inflammatory drug phenylbutazone (PBZ). We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical intermediates of PBZ, UV-vis spectrophotometry to monitor PBZ oxidation, oxygen analysis to determine the involvement of C-centered radicals, and LC/MS to determine the resulting metabolites. Using EPR spectroscopy and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3(•-) (DMPO/(•)OH) was attenuated with increasing PBZ concentrations. The resulting PBZ radical, which was assigned as a carbon-centered radical based on computer simulation of hyperfine splitting constants, was trapped by both DMPO and MNP spin traps. Similar to Cu,Zn-SOD-peroxidase activity, an identical PBZ carbon-centered radical was also detected with the presence of both myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). Oxygen analysis revealed depletion in oxygen levels when PBZ was oxidized by SOD peroxidase-activity, further supporting carbon radical formation. In addition, UV-vis spectra showed that the λmax for PBZ (λ = 260 nm) declined in intensity and shifted to a new peak that was similar to the spectrum for 4-hydroxy-PBZ when oxidized by Cu,Zn-SOD-peroxidase activity. LC/MS evidence supported the formation of 4-hydroxy-PBZ when compared to that of a standard, and 4-hydroperoxy-PBZ was also detected in significant yield. These findings together indicate that the carbonate radical, a product of SOD peroxidase activity, appears to play a role in PBZ metabolism. Interestingly, these results are similar to findings from heme peroxidase enzymes, and the context of this metabolic pathway is discussed in terms of a mechanism for PBZ-induced toxicity.

Polymer strip films as a robust, surfactant-free platform for delivery of BCS Class II drug nanoparticles.

The robustness of the polymer strip film platform to successfully deliver a variety of BCS Class II drug nanoparticles without the need for surfactant while retaining positive characteristics such as nanoparticle redispersibility and fast dissolution is demonstrated. Fenofibrate (FNB), griseofulvin (GF), naproxen (NPX), phenylbutazone (PB), and azodicarbonamide (AZD) were considered as model poorly water-soluble drugs. Their aqueous nanosuspensions, produced via wet stirred media milling, were mixed with hydroxypropyl methylcellulose solution containing glycerin as plasticizer, followed by casting and drying to form films. For the purpose of comparison, sodium dodecyl sulfate (SDS) was used as surfactant, but was found to be unnecessary for achieving fast dissolution (t80 between 18 and 28 min) for all five drugs. Interestingly, SDS was required for the full recovery of nanoparticles for PB, yet lack of it did not impact the dissolution. Interactions between drug and polymer were investigated with FTIR spectroscopy whereas drug crystallinity within the film was investigated via Raman spectroscopy. Films for all drugs, even for very small samples, exhibited excellent content uniformity (RSD <4%) regardless of use of surfactant. Overall, these results demonstrate the novelty and robustness of the polymer strip film platform for fast release of poorly water-soluble drugs without requiring any surfactants.

Synthesis, characterization and the interaction of some new water-soluble metal Schiff base complexes with human serum albumin.

Some new water-soluble Schiff base complexes of Na2[M(L)(H2O)n]; (M=Zn, Cu, Ni, Mn) with a new water-soluble Schiff base ligand where L denotes an asymmetric N2O2 Schiff base ligands; N,N'-bis(5-sulfosalicyliden)-3,4-diaminobenzophenone (5-SO3-3,4-salbenz) were synthesized and characterized. The formation constants of the water soluble Schiff base complexes were calculated by Ketelaar's equation. The theoretical molecular structure for the complexes was computed by using the HF method and the 6-311G basis set. The mechanism of binding of Na2[M(L)(H2O)n] with human serum albumin (HSA) was studied by fluorescence spectroscopic technique. The results of fluorescence titration showed that the intrinsic fluorescence of HSA was quenched by the complexes; which was rationalized in terms of the dynamic quenching mechanism. The values of Stern-Volmer constants, quenching rate constants, binding constants, binding sites and average aggregation number of HSA have been determined. The thermodynamic parameters, were calculated by van't Hoff equation, indicate that the binding is entropy driven and enthalpically disfavored. Based on the Förster theory of non-radiation energy transfer, the efficiency of energy transfer and the distance between the donor (Trp residues) and the acceptor (complex) were obtained. Finally, the growth inhibitory effects of the complexes toward the K562 cancer cell line were measured.

Physico-chemical solid-state characterization of pharmaceutical pyrazolones: an unexpected thermal behaviour.

In this work, the thermal behaviour of three active substances (phenazone, aminophenazone, phenylbutazone) was studied by drawing up the TG/DTG/DTA curves in air/nitrogen atmosphere at 10 °C min(-1) heating rate. The information on the thermal-induced events was corroborated with the IR spectra of the solid samples (pharmaceutical compounds and the remaining chars after heating treatment), respectively with the ones obtained by evolved gases analysis (EGA). The data on a possible drug-excipient interaction were obtained from the thermoanalytical study of mixtures of these active compounds with talc, magnesium stearate, starch and microcrystalline cellulose. No changes were observed by TG/DTG/DTA curves of mixtures in comparison with the pure compound. Even if the three active substances contain the same heterocyclic ring, having similar molecular structures, their thermal behaviour is not similar. According to thermal and evolved gas analysis, it was proved that formation of CO2 does not involve atmospheric oxygen. By stoichiometric means, the molecular breakdown of aminophenazone can generate only carbon monoxide, which undergoes disproportionation, generating CO2.

Diffusion coefficients of phenylbutazone in supercritical CO2 and in ethanol.

The diffusion coefficients D(12) of phenylbutazone at infinite dilution in supercritical CO(2) were measured by the chromatographic impulse response (CIR) method. The measurements were carried out over the temperature range from 308.2 to 343.2 K at pressures up to 40.0 MPa. In addition, the D(12) data of phenylbutazone at infinite dilution in ethanol were also measured by the Taylor dispersion method at 298.2-333.2K and at atmospheric pressure. The D(12) value of phenylbutazone increased from 4.45×10(-10) m(2) s(-1) at 298.2 K and 0.1 MPa in ethanol to about 1.43×10(-8) m(2) s(-1) at 343.2 K and 14.0 MPa in supercritical CO(2). It was found that all diffusion data of phenylbutazone measured in this study in supercritical CO(2) and in ethanol can be satisfactorily represented by the hydrodynamic equation over a wide range of fluid viscosity from supercritical state to liquid state with average absolute relative deviation of 5.4% for 112 data points.

PAMAM dendrimer-drug interactions: effect of pH on the binding and release pattern.

Understanding the dendrimer-drug interaction is of great importance to design and optimize the dendrimer-based drug delivery system. Using atomistic molecular dynamics (MD) simulations, we have analyzed the release pattern of four ligands (two soluble drugs, namely, salicylic acid (Sal), L-alanine (Ala), and two insoluble drugs, namely, phenylbutazone (Pbz) and primidone (Prim)), which were initially encapsulated inside the ethylenediamine (EDA) cored polyamidoamine (PAMAM) dendrimer using the docking method. We have computed the potential of mean force (PMF) variation with generation 5 (G5)-PAMAM dendrimer complexed with drug molecules using umbrella sampling. From our calculated PMF values, we observe that soluble drugs (Sal and Ala) have lower energy barriers than insoluble drugs (Pbz and Prim). The order of ease of release pattern for these drugs from G5 protonated PAMAM dendrimer was found to be Ala > Sal > Prim > Pbz. In the case of insoluble drugs (Prim and Pbz), because of larger size, we observe much nonpolar contribution, and thus, their larger energy barriers can be reasoned to van der Waals contribution. From the hydrogen bonding analysis of the four PAMAM-drug complexes under study, we found intermolecular hydrogen bonding to show less significant contribution to the free energy barrier. Another interesting feature appears while calculating the PMF profile of G5NP (nonprotonated)-PAMAM-Pbz and G5NP (nonprotonated)-PAMAM-Sal complex. The PMF was found to be less when the drug is bound to nonprotonated dendrimer compared to the protonated dendrimer. Our results suggest that encapsulation of the drug molecule into the host PAMAM dendrimer should be carried out at higher pH values (near pH 10). When such complex enters the human body, the pH is around 7.4 and at that physiological pH, the dendrimer holds the drug tightly. Hence the release of drug can occur at a controlled rate into the bloodstream. Thus, our findings provide a microscopic picture of the encapsulation and controlled release of drugs in the case of dendrimer-based host-guest systems.

Binding of diuretic antihypertensive bendroflumethiazide to human serum albumin studied by ¹9F nuclear magnetic resonance method.

Simultaneous specific and nonspecific binding of bendroflumethiazide (BFZ) to human serum albumin (HSA) and concentration profile of BFZ in HSA buffer (pH 7.40) solution were investigated by ¹9F nuclear magnetic resonance (NMR) method. The ¹9F NMR spectrum of BFZ (200 µM) in a buffer solution showed a sharp signal of its CF3 group at 17.8 ppm from the reference trifluoroethanol. Addition of 0.60mM HSA to the sample solution caused the CF(3) signal splitting into three broadened peaks at 18.4 (A), 17.9 (B) and 17.4 ppm (C). By its chemical shift and spectral behavior, B was assigned to unbound BFZ. Competition experiments with Site I and II ligands lead to C being assigned to Site II bound BFZ. However, the peak intensity (areas) of A was not reduced by these ligands, suggesting that A arises from nonspecific binding. Using the peak intensities at several total concentrations of BFZ, Scatchard plot was performed. The plot for A provided a straight line parallel to the x-axis confirming nonspecific binding and that for C was consistent with specific binding. The binding constants for nonspecific and specific Site II binding were 1.02 and 1.00 × 104 (M⁻¹) (n=1.1), respectively. The presence of 0.10 M Cl⁻ in the sample solution affected the binding constant of Site II binding, but not that of nonspecific binding. The concentration profile of BFZ calculated using the binding constants revealed that nonspecific binding is more effective than Site II binding for the binding of BFZ to HSA. It was also confirmed that considerable amounts of BFZ liberated from Site II by the Site II ligands or Cl⁻ ions bind again nonspecifically.

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases.

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ((1)HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants K(a) were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants K(aI) of PBZ-SA complex. Similarly, PBZ influences K(aI) of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of K(aII) values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of (1)HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

Physicochemical characterisation, drug polymer dissolution and in vitro evaluation of phenacetin and phenylbutazone solid dispersions with polyethylene glycol 8000.

Poor water solubility leads to low dissolution rate and consequently, it can limit bioavailability. Solid dispersions, where the drug is dispersed into an inert, hydrophilic polymer matrix can enhance drug dissolution. Solid dispersions were prepared using phenacetin and phenylbutazone as model drugs with polyethylene glycol (PEG) 8000 (carrier), by melt fusion method. Phenacetin and phenylbutazone displayed an increase in the dissolution rate when formulated as solid dispersions as compared with their physical mixture and drug alone counterparts. Characterisation of the solid dispersions was performed using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). DSC studies revealed that drugs were present in the amorphous form within the solid dispersions. FTIR spectra for the solid dispersions of drugs suggested that there was a lack of interaction between PEG 8000 and the drug. However, the physical mixture of phenacetin with PEG 8000 indicated the formation of hydrogen bond between phenacetin and the carrier. Permeability of phenacetin and phenylbutazone was higher for solid dispersions as compared with that of drug alone across Caco-2 cell monolayers. Permeability studies have shown that both phenacetin and phenylbutazone, and their solid dispersions can be categorised as well-absorbed compounds.

Assessment of diffuse transmission and reflection modes in near-infrared quantification, part 2: DIFFuse reflection information depth.

Near-infrared spectroscopy offers tremendous advantages for pharmaceutical manufacturing as a fast and nondestructive method of quantitative and qualitative analysis. Content uniformity (end-product analytics) and process analytics are two important applications of the method. Diffuse reflection (DR) information depth (vertical sampling span) assessment is of equal importance in content prediction applications and to understand the effect of inhomogeneities in the sample. Three experiments were conducted: (a) 0.5 to 10.0 mm incremental thickness MCC tablets with constant porosity, (b) MCC/phenylbutazone (PBZ) double-layered (DL) tablets (PBZ layer 0%-100% in 0.5 mm steps), and (c) Comparison of placebo and 30% caffeine tablet cores with incremental film coating (film thickness of 0-0.35 mm). Incremental thickness and cluster analysis of DL tablets showed that DR information depth was <0.5 mm, whereas the data fitting from incremental coating showed that signal drop reached 50% at 0.05 to 0.07 mm, depending on the wavenumber and 90% signal drop (10% information content) can be seen between 0.20 and 0.25 mm without extrapolation. These results mean that DR mode for pharmaceutical tablets obtains spectral information from the very surface, and radiation is barely reflected back from beyond thin-film coatings, making it less useful than diffuse transmission mode for core content analysis, especially for thick-coated, multilayer, multicore, or highly inhomogeneous tablets.

Surveillance study of novobiocin and phenylbutazone residues in raw bovine milk using liquid chromatography-tandem mass spectrometry.

A simple method permitting the simultaneous determination of trace residues of novobiocin and phenylbutazone in raw milk samples using liquid chromatography-tandem mass spectrometry was developed. Raw milk samples were mixed with acetonitrile to facilitate the concurrent precipitation of milk proteins and extraction of both veterinary drugs. Without additional clean-up or concentration of the resulting extract, the analytes could be quantified at concentrations as low as 0.0025 and 0.001 microg ml(-1) for phenylbutazone and novobiocin, respectively. The analysis of a series of fortified raw milk samples at analyte concentrations ranging from 0.005 to 0.1 microg ml(-1) and from 0.01 to 0.2 microg ml(-1) for phenylbutazone and novobiocin, respectively, yielded average recoveries ranging from 89.2% to 104.3% with standard deviations below 7%. The analytical method was applied to the analysis of raw milk samples collected from transport trucks upon delivery at dairy-processing plants throughout Alberta, Canada. Novobiocin was detected in 13 of 1072 samples tested at concentrations ranging from 0.001 to 0.007 microg ml(-1). Phenylbutazone was not detected in any of the samples tested.

Host-guest chemistry of dendrimer-drug complexes. 3. Competitive binding of multiple drugs by a single dendrimer for combination therapy.

The host-guest chemistry of dendrimer-drug complexes is of great significance to the design and optimization of dendrimer-based drug delivery systems. The competitive binding of multiple drugs by a single dendrimer in aqueous solutions was investigated by (1)H NMR and 2D-NOESY studies. These rapid, noninvasive, and accurate NMR techniques allow us to monitor the signals of various drugs as well as carriers in a complicated host-guest system. Ethanol was used as an internal standard to simultaneously quantify dendrimers and drugs and to estimate the binding ability of dendrimers toward different drug molecules. The results suggested that supramolecular structure of dendrimer-multiple drug complexes is formed based on electrostatic interactions and hydrophobic/hydrogen-bond interactions. Factors including hydrophobic properties, sizes, pK(a) values, charged groups, and spatial hindrance effects of the drugs influenced the localization of drug molecules on the surface and in the interior pockets. In a ternary host-guest system of dendrimer/mycophenolic acid/phenylbutazone, many more phenylbutazone molecules localized in the inner pockets than mycophenolic acid, while more mycophenolic acid bound on the surface by ion-pairs than phenylbutazone. These results provide new insight into host-guest chemistry of dendrimer-drug complexes and the design/optimization of dendrimer-based drug delivery systems.