Evaluating the Capacity of Human Serum Albumin to Reduce Non-Specific Binding of Meloxicam in the Ultrafiltration Process

Abstract

Due to the importance that plasma protein binding of drugs plays and the constant interest to optimize study methods of the process, this study aimed to test the capacity of a human serum albumin solution to reduce non-specific binding of highly bound meloxicam, based on the ability of the protein to block most non-specific binding sites of the ultrafiltration devices. Samples of meloxicam and internal standard piroxicam were prepared in both phosphate-buffered saline solution, commonly used in biological research, and 5% human serum albumin solution. After the ultrafiltration process, the free drug fraction was analysed using a reversed phase HPLC-UV method. A decrease in the concentration of meloxicam after ultrafiltration in both matrices was observed, the decrease being higher in the case of phosphate-buffered saline solution. The mean determined degree of binding to albumin for meloxicam was 55%. The high decrease in concentration for meloxicam in the non-proteic matrix indicates a very high degree of non-specific binding, phenomenon which appears to be indeed reduced for the samples prepared in human serum albumin solution. The study also emphasises the very close attention that should be paid to the preparation and processing of samples in protein matrices and to the experimental conditions. RezumatDatorită importanței pe care legarea medicamentelor de proteinele plasmatice o prezintă și a interesului constant de a optimiza metodele de studiu ale procesului, acest studiu a avut ca scop testarea capacității unei soluții de albumină serică umană de a reduce legarea nespecifică a meloxicamului puternic legat, pe baza capacității proteinei de a bloca majoritatea situsurilor de legare nespecifică a dispozitivelor de ultrafiltrare. Probele de meloxicam și standard intern piroxicam au fost preparate atât în soluție de tampon fosfat salin, utilizată în mod obișnuit în cercetări biologice, cât și în soluție de albumină serică umană 5%. După procesul de ultrafiltrare, fracția de medicament liber a fost analizată folosind o metodă HPLC-UV în fază inversă. S-a observat o scădere a concentrației de meloxicam după ultrafiltrare în ambele matrici, scăderea fiind mai mare în cazul soluției de tampon fosfat salin. Gradul mediu de legare de albumină determinat pentru meloxicam a fost de 55%. Scăderea ridicată a concentrației de meloxicam în matricea non-proteică indică un grad foarte ridicat de legare nespecifică, fenomen care pare a fi într-adevăr redus pentru probele preparate în soluție de albumină serică umană. Studiul scoate în evidență, de asemenea, atenția mare care trebuie acordată condițiilor experimentale, preparării și prelucrării probelor în matrici proteice.

Enantioselective binding of carvedilol to human serum albumin and alpha‐1‐acid glycoprotein

“…Regarding the main known disadvantage of the method represented by the nonspecific binding (NSB) of drugs to parts of the UF device, we considered it to be negligible in our study, taking into consideration the literature reports made by Wang and Williams, who have shown that even in the case of highly lipophilic compounds, NSB is not a limitation when drugs are incubated with the UF device in protein matrices because of their ability to block the NSB sites 37 . This theory was also tested and proven in our previous experience 38 …”

“…37 This theory was also tested and proven in our previous experience. 38 The concentrations of carvedilol for the binding assays ranges were chosen taking into account the maximum plasma concentrations detected in patients reported in previous studies. Depending on the dose regimen and type of pharmaceutical formulation administered (immediate release, controlled release), a great variability regarding the maximum plasma concentration was often observed with ranges that could go from a value below 7 ng/mL to a value over 350 ng/mL.…”

Comparison of Different Ultrafiltration Devices for the Study of Plasma Protein Binding of Carvedilol

“…According to the data sheet of the product, only the Centrifree devices were specifically designed for separating free from bound microsolutes in biological samples, but other PPB studies report good results also obtained using the Amicon Ultra devices, initially designed for concentration of different components of biological samples (antigens, antibodies, enzymes, nucleic acids, microorganisms), protein extraction and purification (Du et al, 2014;Imre et al, 2021).…”