Protein Binding Drug-Drug Interaction between Warfarin and Tizoxanide in Human Plasma

Abstract

The goal of the in vitro research was to evaluate the potential for inhibition of warfarin protein binding by tizoxanide. Warfarin was of particular interest for the present investigation because it has been shown to be highly bound to plasma proteins and is a narrow therapeutic index drug. Tizoxanide is an active metabolite of an anti-infective prodrug nitazoxanide and also highly protein-bound medication. Both drugs are expected to be co-administered clinically. Protein binding of warfarin was investigated using a centrifugal ultrafiltration technique. Co-administration of tizoxanide significantly inhibited protein binding of warfarin for all concentrations tested. Tizoxanide increased free fraction (f_u) of warfarin 34, 28, and 20-fold for concentration of 50, 75, and 100 μg/ml, respectively. The interaction could potentially result in increasing the toxicity of warfarin therapy and the risk of bleeding.

Keywords: Protein binding; Interaction; Warfarin; Tizoxinide

Introduction

Many drugs are bound to proteins within the body. There is marked variability in the extent to which the drugs bind to the proteins. The degree of drug-protein binding might affect a drug’s efficiency. Drugs can be bound to plasma proteins as well as tissue proteins. Plasma protein binding can have multiple effects on the pharmacokinetics (PK) of a drug. The binding of drug to plasma proteins is a major determinant of drug disposition. When a drug is bound to the plasma proteins, it is not actively distributed to the site of action to interact with the target tissues. Moreover, the bound drug is kept in the blood, while the unbound (free) fraction may be metabolized or excreted. The binding has a very important effect also on drug pharmacodynamics (PD). Only the free drug interacts with receptors, therefore only the free drug can produce therapeutic effect. Common blood proteins that bind drugs are human serum albumins, lipoproteins, glycoproteins, and globulins. Albumin is the most abundant protein in the blood plasma. Many medications extensively bind to the albumins.

Protein binding interactions are displacement reactions, which have been implicated as the causative mechanisms in many drug-drug interactions. Competitive displacement interactions are predominant ones among protein binding reactions. The competitive protein binding drug-drug interactions result in increased the free plasma concentrations of the displaced medications. Clinically displacement reactions acquire importance when the displaced drug is highly bound to plasma proteins. In this case the displacer drug produces a very rapid and significant increase in the plasma concentrations of the displaced medication. The result of this interaction can be dramatic if the second drug is a drug with narrow therapeutic index. Such type of interactions can lead to enhanced pharmacological effects and, possibly, toxicity of the displaced medication.

A simple, generalized guideline for the evaluation of the clinical significance of protein binding interactions frequently cannot be applied. Instead, a careful analysis of protein binding effects must be made on a drug-by-drug basis. Free, unbound concentrations should always be measured rather than estimated, and used as the primary input for PK/PD correlations, as well as for statements on the safety and efficacy of drugs [1].

Warfarin (Coumadin™) is a widely used anticoagulant. Warfarin is indicated for treatment of venous thromboembolism, atrial fibrillation, mechanical and bioprosthetic heart valves, post-myocardial infarction, and recurrent systemic embolism. Warfarin consists of a racemic mixture of two active enantiomers: R- and S-forms [2,3]. Warfarin is a narrow therapeutic range drug and can cause major or fatal bleeding. Approximately 99% of the drug is bound to human plasma albumins [3]. One of the pharmacokinetic mechanisms for drug interactions with warfarin is protein binding interaction. This interaction can result in displacement of warfarin from the protein binding site, increase warfarin free plasma concentration, and increase the risk of warfarin toxicity. Free drug plasma concentrations must be carefully monitored during warfarin therapy.

Nitazoxanide (Alinia™) is an anti-infective prodrug, which converts to an active metabolite tizoxanide very fast. The parent nitazoxanide is not detected in plasma. Tizoxanide is active against anaerobic bacteria, protozoan parasites, and viruses [4,5]. Tizoxanide is highly bound to plasma proteins (>99 %) [4,6].Caution should be used when administering nitazoxanide concurrently with other highly plasma protein-bound drugs, especially drugs with narrow therapeutic indices, as competition for binding sites may occur.

Tizoxanide is compound that is expected to be co-administered with warfarin clinically and have the potential to alter warfarin protein binding. No drug-drug protein binding interaction studies have been reported for warfarin and tizoxanide.

The overall goal of the research was to evaluate the potential for inhibition of protein binding of warfarin by tizoxanide, which can be expected to be co-administered with warfarin in the patients.

Materials and Methods

Materials

Warfarin, DMSO, acetonitrile and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Tizoxanide was purchased from Cayman Chemical (Tallinn, Estonia). HPLC nylon filter (0.45 micron, 47 mm) was purchased from Pall Life Science (Port Washington, NY). Human plasma was purchased from Valley Biomedical (Winchester, VA). Amicon Centrifree YM-30 (molecular weight cut off 30K) centrifugal filter devices were obtained from Millipore Corporation (Billerica, MA, USA).

Study groups

The studies were divided into two phases. Four experiments were performed for each study group. Phase I studies generated baseline protein binding values for warfarin. The compound was dosed alone. Phase II studies was conducted to assess the effect of tizoxanide on warfarin protein binding. Warfarin and tizoxanide were dosed together. Tizoxanide was selected based on its potential to be co-administered with warfarin in the clinical setting as well as its protein binding profile. Warfarin protein binding data of Phase I studies were compared to the warfarin binding values of Phase II studies. Warfarin concentrations were determined by HPLC.

Analytical method for warfarin

Warfarin was measured in all samples by validated HPLC method. Separation was accomplished using a C18 column (100 mm X 4.6 mm, 2.6μ, and 100A°) with a pre-column filter. The C18 column was temperature controlled at 30°C. Measurements were performed with ultraviolet detection at a wavelength of 220 nm. Mobile phase consisted of 35% acetonitrile and 65% 10 mM phosphate buffer (KH₂PO₄, pH 6.5). Analysis was conducted at ambient temperature at a constant flow rate of 1.0 mL/min using isocratic elution. Six microliters of the resultant ultrafiltrate was injected into the HPLC.

Binding assessments

Nonspecific binding to ultra filtration device: A preliminary study was performed to identify warfarin nonspecific binding to the ultrafiltration device. Aqueous solution of warfarin was added to an ultrafiltration device (Amicon Centrifree Micropartition System®). The system was centrifuged for 15 minutes. The ultrafiltrate was assayed to determine warfarin binding to the ultrafiltration system. Samples were analyzed by HPLC. The studies were performed in triplicate.

Protein binding determination: Plasma protein binding of warfarin was evaluated using ultrafiltration. Studies were carried out at physiologic temperature and pH. Aliquots of human plasma were placed in test tubes and spiked with stock solutions of warfarin to generate samples of varying concentrations (e.g., 50, 75, and 100 μg/ml). Tizoxanide (500 μg/mL) was added only for interaction studies (Phase II). Samples were allowed to equilibrate in shaker bath (37°C) for 30 minutes. An aliquot (1 ml) of warfarin human plasma was added to an Amicon Centrifree Micropartition System® (MW cutoff 30,0000). The system was centrifuged for 15 minutes @ 1500 g. The ultrafiltrate was assayed for determination of free warfarin concentrations. Samples were analyzed by HPLC. All experiments were performed in quadruplicate.

Data analysis

Unbound fraction (f_u) of warfarin was calculated as a ratio of unbound (ultrafiltrate) concentration and total plasma concentration.

f_u = C_u/C_t

Where: f_u is unbound (free) fraction

C_u is unbound (free) concentration of warfarin

C_t is total concentration of warfarin

Statistical analysis

Mean estimates of protein binding parameters were compared using ANOVA. In this manner, differences in protein binding of warfarin were detected between study groups. Statistical significance was taken as a p-value <0.05.

Results(NMS)

Protein binding of warfarin was investigated using a centrifugal ultrafiltration technique. Experiments were conducted over a range of warfarin concentrations from 50 to 100μg/mL. Preliminary studies (i.e., fraction unbound ~ 1) found negligible binding of warfarin to the ultrafiltration device (Amicon Centrifree™ YM-30 micropartition devices). Warfarin protein binding data presented in Table 1.

  

    
Study Groups 
    
Warfarin Concentrations (μg/mL) 
    
Fraction Unbound of Warfarin* 
  
  

    
Phase I:
      
Warfarin alone
    
50
    
0.010 (0.003)
  
  

    
75
    
0.011 (0.002)
  
  

    
100
    
0.014 (0.001)
  
  

    
Phase II:
      
Warfarin + Tizoxanide
    
50
    
0.339 (0.024)
  
  

    
75
    
0.313 (0.008)
  
  

    
100
    
0.277 (0.061)
  

Table 1:  Protein Binding of Warfarin.