UPLC-based assay to assess the hydrophobicity of Antibody-Drug Conjugate (ADC) payloads.

Antibody-Drug Conjugates (ADCs) consist of antibodies attached to cytotoxic small molecules or biological agents (i.e., payloads) through chemical linkers which may be cleavable or non-cleavable. The development of new ADCs is challenging, particularly the process of attaching the linker-payload construct to the antibody (i.e., the conjugation process). One of the major problems associated with conjugation is high hydrophobicity of the payload which can lead to low yields of the ADC through aggregation and/or lower than desired Drug-Antibody Ratios (DARs). We report here a UPLC-based assay that can be used to study the physicochemical properties of ADC payloads at an early stage of development, and to provide information on whether the hydrophilic-hydrophobic balance is suitable for conjugation or further physicochemical optimization is required. The assay is relatively simple to establish and should be of use to those working in the ADC area.

Novel spectral manipulations for determinations of Tolnaftate along with related toxic compounds: Drug profiling and a comparative study.

A comparative study using novel quadruple divisor and mean centering of ratio spectra spectrophotometric methods was developed for resolution of five- component mixture of Tolnaftate, ß-naphthol (Tolnaftate alkaline degradation product and its toxic impurity), methyl(m-tolyl)carbamic acid (Tolnaftate alkaline degradation product), N-methyl-m-toluidine (Tolnaftate toxic impurity) and methyl paraben (as a preservative). For the novel quadruple divisor method, each component in the quinary mixture was determined by dividing the quinary mixture spectrum by a sum of standard spectrum of equal concentration of the other four components as a quadruple divisor. First derivative of each ratio spectra was then obtained which allowed selective determination of each component without interference from other components in the mixture. The second method was mean centering of ratio spectra that depended on utilizing the mean centered ratio spectra in four successive steps leading to enhancement of the signal to noise ratio. The absorption spectra of the five studied components were recorded in the wavelength range of 210-350 nm. The mean centered fourth ratio spectra amplitudes for each component were used for its determination. The developed methods were successfully applied for determination of laboratory prepared quinary mixtures to ensure method's specificity, then, were further applied on Tinea Cure® cream where no interference from excipients. For the first time, Tolnaftate was determined along with its toxic impurity; ß-naphthol, that could be absorbed by the skin, causing systemic toxic effects, unlike Tolnaftate that poorly absorbed, indicating the significance of this work. The proposed methods were statistically compared with each other and with the reference method. Furthermore, ICH guidelines were followed for their validation.

Enhancement of the topical tolnaftate delivery for the treatment of tinea pedis via provesicular gel systems.

Tolnaftate is a thiocarbamate antifungal drug which is therapeutically active against dermatophytes that cause various forms of tinea. Due to the small amount of tolnaftate released from ordinary ointment bases and insufficient penetration through the infected skin layers the need to incorporate the drug in a more suitable pharmaceutical form has evolved. A provesicular system is one such form that can solve these problems. Once in contact with the skin, dilution with moisture occurs and the provesicular system rapidly transforms into a vesicular one. Provesicular systems were prepared according to full-factorial experimental design. Plain provesicular systems were compared with systems containing Phospholipon 80 H and Lipoid S45 as penetration enhancers. Design expert software was used to analyze the effect of formulation variables (type of Span used as well as the presence or the absence of the penetration enhancer and its type) on the dependent variables: percent encapsulation efficiency (EE%), vesicle size and percent in vitro drug released). Three formulations were chosen; a plain provesicular system (PV-2), one containing Phospholipon 80H (PV-6) and another containing Lipoid S45 (PV-10) with the goal to reveal the effect of penetration enhancer on morphology, rheological properties and ex vivo permeation using confocal laser scanning microscopy (CLSM). Analysis of CLSM results showed that the penetration enhancing effect for the tested formulations followed the order PV-10 > PV-6 > PV-2. Promising clinically active treatment for tinea patients could be expected as shown by the in vivo permeation results for the provesicular systems as suggested by the CLSM results.

Detailed study of precipitation of a poorly water soluble test compound using methodologies as in activity and solubility screening - mixing and automation effects.

Storage of pharmaceutical discovery compounds dissolved in dimethylsulfoxide (DMSO) is commonplace within industry. Often, the DMSO stock solution is added to an aqueous system (e.g. in bioassay or kinetic solubility testing)- since most test compounds are hydrophobic, precipitation could occur. Little is known about the factors affecting this precipitation process at the low (µM) concentrations used in screening analyses. Here, a poorly water soluble test compound (tolnaftate) was used to compare manual and automated pipetting, and explore the effect of mixing variables on precipitation. The amount of drug present in the supernatant after precipitation and centrifugation of the samples was quantified. An unusual result was obtained in three different laboratories: results of experiments performed initially were statistically significantly higher than those performed after a few days in the same lab. No significant differences were found between automated and manual pipetting, including in variability. Vortex mixing was found to give significantly lower supernatant amounts compared to milder mixing types. The mixing employed affects the particle growth of the precipitate. These findings are of relevance to discovery stage bioassay and kinetic solubility analyses.

Study of tolnaftate release from fatty acids containing ointment and penetration into human skin ex vivo.

Five fatty acids (oleic, linoleic, myristic, lauric and capric) were incorporated in 10% (w/w) into ointment formulation and their influence on lipophilic model drug tolnaftate release in vitro and enhancing effect on tolnaftate penetration into epidermis and dermis of human skin ex vivo were investigated. The prepared ointments were tested for homogeneity, pH and theological properties. In vitro release studies and ex vivo skin penetration experiments were carried out using Hanson and Bronaugh-type flow-through diffusion cells, respectively. Tolnaftate cumulative amount liberated from semisolids was assayed using UV-Vis spectrophotometer. After in vitro skin penetration studies, appropriately extracted human skin layers were analyzed for tolnaftate content using a validated HPLC method. Statistical analysis revealed that release rate of tolnaftate from control ointment and ointments with fatty acids was not significantly different and only 7.34-8.98% of drug was liberated into an acceptor medium after 6 h. Tolnaftate amount penetrating into 1 cm2 of epidermis from ointments containing oleic, linoleic, myristic and lauric acids was significantly greater (p < 0.05) than from the control ointment. Penetration enhancing ratios for these fatty acids for tolnaftate penetration into epidermis ranged from 1.48 to 1.75. In conclusion, fatty acids did not increase the liberation of tolnaftate from ointment formulation, but demonstrated their enhancing effect on tolnaftate penetration into human epidermis in vitro. Results from in vitro release experiments do not suit for prediction of the situation in the skin in vitro, if chemical penetration enhancers are incorporated into the ointment formulation.

Nonplanar property study of antifungal agent tolnaftate-spectroscopic approach.

Vibrational analysis of the thionocarbamate fungicide tolnaftate which is antidermatophytic, antitrichophytic and antimycotic agent, primarily inhibits the ergosterol biosynthesis in the fungus, was carried out using NIR FT-Raman and FTIR spectroscopic techniques. The equilibrium geometry, various bonding features, harmonic vibrational wavenumbers and torsional potential energy surface (PES) scan studies have been computed using density functional theory method. The detailed interpretation of the vibrational spectra has been carried out with the aid of VEDA.4 program. Vibrational spectra, natural bonding orbital (NBO) analysis and optimized molecular structure show the clear evidence for electronic interaction of thionocarbamate group with aromatic ring. Predicted electronic absorption spectrum from TD-DFT calculation has been compared with the UV-vis spectrum. The Mulliken population analysis on atomic charges and the HOMO-LUMO energy were also calculated. Vibrational analysis reveals that the simultaneous IR and Raman activation of the C-C stretching mode in the phenyl and naphthalene ring provide evidence for the charge transfer interaction between the donor and acceptor groups and is responsible for its bioactivity as a fungicide.

Study on the supramolecular multirecognition mechanism of beta-naphthol/beta-cyclodextrin/anionic surfactant in a tolnaftate hydrolysis system.

Based on the fact that tolnaftate degrade to beta-naphthol sodium (RONa) at 5.00 mol/L NaOH solution and RO(-) was protonated to ROH after being acidified and adjusted to the pH 4.50 by acetic acid-sodium acetate buffer solution, we studied and discussed the mechanism of the supramolecular multirecognition interaction among the anionic surfactants sodium lauryl sulfate (SLS), beta-cyclodextrin (beta-CD), and beta-naphthol (ROH) by means of fluorescence spectrum, surface tension of the solution, infrared spectrograms, and (1)HNMR spectroscopy. The apparent formation constant of the ternary inclusion complex was determined to be (5.48 +/- 0.13) x 10(3) L(2)/mol(2). The thermodynamic parameters (DeltaG degrees, DeltaH degrees, DeltaS degrees ) for the formation of the inclusion complexes were obtained from the van't Hoff equation. It was indicated that the multiple and synergistic protection effect of SLS and beta-CD on the excited singlet state ROH played very important roles in the enhancement of the fluorescence of ROH. Results showed that, at room temperature, the naphthalene ring of ROH and the hydrophobic hydrocarbon chain of SLS were included into the cavity of beta-CD to form a ROH/SLS/beta-CD ternary inclusion complex with stoichiometry of 1:1:1, which provided effective protection for the excited state of ROH and increased the fluorescent intensity of ROH obviously.

X-ray powder diffractometric method for quantitation of crystalline drug in microparticulate systems. I. Microspheres.

Ethylcellulose microspheres containing tolnaftate (I) were prepared by the emulsion-solvent evaporation technique. An X-ray powder diffractometric method was developed to quantify the content of crystalline I in these microspheres. X-ray lines of I with d-spacings of 5.5 and 4.2 A were chosen for the quantitative analyses. Physical mixtures containing various weight fractions of I and blank (empty) microspheres were prepared and lithium fluoride (20% w/w) was added as the internal standard. The 5.5 and 4.3 A lines of I and the 2.3 A line of lithium fluoride were used for the quantitative analysis. A plot of the intensity ratio (intensity of the 5.5 A line of I/intensity of 2.3 A line of lithium fluoride) as a function of the weight percent of I in the mixture, resulted in a straight line. The crystalline content of I in the tolnaftate-loaded microspheres was determined using this standard curve. A second independent determination of the content of I was possible from the intensities of the 4.3 A line. The enthalpy of fusion of I, determined by differential scanning calorimetry (DSC), was also used as a measure of the crystalline content of I in the microspheres. The X-ray and DSC methods measure the content of crystalline I in the microspheres at room temperature ( approximately 25 degrees C) and at the melting point of I (111 degrees C), respectively. The total content of I in the microspheres was determined by HPLC. The DSC and X-ray results indicated that a substantial fraction of the incorporated I was dissolved in the ethylcellulose matrix.

Determination of the physical state of drug in microcapsule and microsphere formulations.

Tolnaftate microcapsules and microspheres were prepared by gelatin-acacia coacervation and emulsion-solvent evaporation methods respectively. The physical state of the drug in these formulations was determined by using scanning electron microscopy (SEM), X-ray powder diffractometry, and differential scanning calorimetry (DSC). High pressure liquid chromatographic (HPLC) method was used for stability determination and polymer-drug interactions were evaluated using FTIR. The pros and cons of each method, in the assessment of the physical state of drug in these formulations, were investigated. SEM was found to be useful in obtaining a direct visual evidence of the presence of crystalline drug in the microspheres, but not for the microcapsule formulation. The DSC method was used to determine the physical state of the drug qualitatively in both these formulations. In the case of the microcapsules, accurate quantitation of the crystalline drug content by DSC was not possible because of the interference of thermal events. Powder X-ray diffractometric method was able to demonstrate the presence of crystalline drug and polymorphic changes, if any, in both these formulations. HPLC data revealed that the drug was stable in these formulations for at least 6 months. The FTIR studies indicated the absence of any drug interaction with the polymeric matrix materials, during preparation of these dosage forms.