INTRODUCTION:

The acid dissociation constant (pKa) indicates the ionization state of a compound at a given pH. This state plays a major role in the absorption, distribution, metabolism, and excretion (ADME) profile of a given compound^1-2. Therefore, the pKa value influences psychochemical properties like: pH dependent aqueous solubility, protein interaction and membrane permeability (2). Hence the pKA value a critical factor for understanding the behavior of a drug inside the body.

Carvedilol (Figure 1) is a nonselective alpha-1 blocker and beta blocker, currently used in clinical practice. It contains antihypertensive properties and is used to treat heart failure, high blood pressure, and angina pectoris³. It may also reduce risk of death following myocardial infarction. The molecular formula of Carvedilol is C₂₄H₂₆N₂O₄and it has a Mw of 406.482. Carvedilol is considered to be a weak base and exhibits pH-depended solubility^3-4.

In its ionized form, Carvedilol is soluble in acidic pH (the stomach), but may precipitate at alkaline pH (Intestine). Carvedilol is poorly soluble in water, which results in a low bioavailability⁴. Due to its poor aqueous solubility, the significance of pKa becomes even higher, especially in the context of its ADME properties⁵.

Figure 1: Carvedilol structure

In previous reports, the pKa value of Carvedilol was estimated and values between 7.7 and 8.8 were determined. According to the open chemistry database Pubchem, the pKa value is 8.77, which is based on the chemical calculator SPARC⁶. Marques et al, reported a pKa of 8.25 and Hoffman et al, reported a value of 7.8^7-9. Since it is essential to have proper knowledge of Carvedilol and its ADME profile, re-evaluating the pKa may be necessary to understand the controversies in its determined pKa value. For this, we use the well established method of Uv-Vis spectroscopy due to its availability, accuracy, simplicity and reproducibility. This is combined with pH titrations, to obtain a sigmoid shaped curve from with the pKa can be estimated from the inflection point and curve fitting^10-11.

MATERIAL AND METHODS:

Drugs and Chemicals:

The Carvedilol compound was obtained as a gift sample from Mylan Laboratories Ltd., Hyderabad, India. Buffers, pH 2-12, were prepared according the UPS and AAT Bioquest protocols. The desired pH value was achieved by increasing or decreasing the buffer pH using 1M HCl or NaOH. For pH2 hydrochloric acid buffer (potassium chloride, Nice chemicals, Kochi – 682024, India, Cat. No. P11829), pH3 a glycine buffer (Glycine, RanBaxy, Mohali - 160055, India, Cat.No G0080) pH 4and5 a Sodium acetate buffer (Sodium acetate, CDH, New Delhi – 110002, India, Cat. No. 030104) 6 and 7 a Phosphate buffer (potassium phosphate, Merck, Mumbai – 400018, India, Cat.No MC3M630067) pH8, 9 and 10 an alkaline borate buffer (boric acid, Fisher, Chennai – 600024, India, Cat.No 6005) and for pH11 and 12 a disodium phosphate buffer (disodium phosphate, HiMedia, Mumbai – 400086, India, Cat.No. RM1257-500G) was used. The pH values were verified using a pH meter and variation was kept within ± 0.05 pH units.

Stock solutions:

A stock solution of 30mM Carvedilol in pure HPLC graded methanol (Finair limited, Ahmedabad – 382110, India, Cat. No. 67-56-1) was prepared. The stock solution was diluted 20 times to obtain a 1.5mM working solution. For measurements, the Carvedilol working solution was diluted 100 times to a final concentration of 15uM in the respective solvents.

Instruments:

Digital pH meter MK VI, Systronics Limited, Ahmedabad – 380013, India. UV-Vis spectrophotometer of model UV-2450, Shimadzu Analytical, New Delhi – 110082, India, Cat. No. 208-24301-93.

UV-Vis measurements:

UV-Vis spectroscopic measurements were performed at in 1ml quartz cuvettes at 30±2ºC. UV-Measurements were carried out in triplicate on 3 different days using freshly prepared stock solutions. For absorption measurements, 241 nm was selected. Non-linear curve fitting (using the Boltzmann sigmoidal algorithm) was performed using Graphpad – Prism 6 (Graphpad Software, California, San Diego, USA)).

RESULTS:

pKa determination by spectrometric titration may be used for any compound, given there is an analytical wavelength at which the ionized form absorbs strongly while the unionized form absorbs lesser, or vice versa. In figure 2, the UV-spectra of carvedilol in neutral buffer (pH 7) is represented. Based on this, 241nm was selected as analytical wavelength. To determine the pKa value of Carvedilol, spectroscopic titrations, in the pH range off 2 to 12, were performed.

Figure 2: UV Spectra of Carvedilol in neutral buffer.

This resulted in a sigmoid shaped pattern. We assumed a Gaussian distribution of the data, thus potential outliers were calculated and excluded using the Graphpad – Prism ROUT method, with a strength of Q5%. The average values were non-linear fitted using the Boltzmann sigmoidal algorithm (figure 3).

Figure 3: Boltzmann sigmoidal algorithm

The error bars indicate the standard error of the mean (SEM) and the dashed lines display the 95% confidence interval. From the inflection point (V50) the pKa value of Carvadilol was estimated to be exactly 7.77 ± 0.09 (rounded to 7.8). To calculate the quality of the fit, both the R²(0.99) as well as the residuals were determined (figure 4).

Figure 4: Calculation of quality of fit

All values remain within ± 0.01 units variation of the calculated sigmoid curve. To ensure the values are evenly distributed and within fitting guidelines, the residuals were linear fitted. The fit (red) showed no deviation from zero, which proves that our fit model is accurate and well suitable for our data.

DISCUSSION:

The pKa of weak base Carvedilol shows controversy in literature. Values between 7.8 and 8.7 have been previously reported. Since the pKa of Carvedilol is key in understanding its ADME profile, knowing the pKa is of major importance. In this article, we aim to re-evaluate the pKa of Carvedilol. To achieve this Uv spectroscopy was used, due to its availability, accuracy, simplicity and reproducibility¹⁰. UV-measurements at 241 nm in buffers ranging from pH 2-12 resulted in a sigmoid shaped pattern. UV-measurements were repeated multiple times in triplicate at different days and carried out by different members of our research group. The measurements at different day showed deviation up to 0.100 units (displayed as SEM). Likely due to fresh stock preparations, since triplicates on the same day displayed only small instrumental variation. We were able to fit the average of all measurements with high correlation (R²=0.99) using the Boltzmann sigmoidal algorithm¹².The residuals of the fit show little deviation from the actual fit, and quality was ensured by linear fitting, which showed no deviation from zero. According to our results, Carvedilol has a pKA value of 7.8 (figure 2). This value is similar to previously described value of 7.8 ^[8] and only differs a little from 8.25 ^[7]. Marques et al, used Raman spectroscopy at 20ºC and as a solvent DMSO. Since the pKa is influenced by factors like temperature and ionic strength, these experimental differences likely explain the small variation in result.¹¹ Furthermore, they measured in a pH range of 2.5-13.5 and it appears the focus is on the secondary amine group only⁷. Our result were different from the by SPARC calculated value of 8.77⁶. Since the SPARC is a prediction and not an experimentally validated value, our Carvedilol pKa determination of 7.8 is considered to be an accurate representation of the pKa value. Due to the present pKa confirmation, the experimental pKa value of Carvadilol could be considered for incorporation in the NCBI PubChem database.

CONCLUSION:

The pKa of Carvedilol was estimated from the inflection point of the Boltzmann-sigmoidal algorithm, which is 7.8. This UV-spectrophotometric method was found to be simple, accurate and can be easily applied to determine the pKa.

ACKNOWLEDGEMENT:

The authors sincerely thank Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, India and Radboud University, the Netherlands for providing necessary facilities to carry out this work.

REFERENCES:

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Received on 21.05.2020 Modified on 01.07.2020

Research J. Pharm. and Tech. 2021; 14(5):2714-2716.

DOI: 10.52711/0974-360X.2021.00478