The History Of The Combination Therapy

Published Date: 02 Nov 2017

Abstract

Background

Plasma protein binding is a major factor in determining the therapeutic profile of a drug. It affects the efficacy of highly protein-bound drugs such as chlorpromazine and verapamil. Verapamil has been used as an adjunct to chlorpromazine in treating severe mania. Detailed research on individual protein binding has been conducted. However, the binding chemistry between proteins and two drugs in combination remains unknown.

Aims

The aim of this study is to evaluate individual and mutual protein binding of chlorpromazine and verapamil to bovine serum albumin (BSA). This project may help provide an insight into whether drug-drug interaction may result in co-administration of chlorpromazine and verapamil. It may help inform clinical guidance in determining dosage regimen to optimize the therapeutic outcome in managing severe mania.

Methods

Individual and mutual protein binding studies of chlorpromazine and verapamil to BSA were conducted using equilibrium dialysis. The degree of protein binding of each drug was assessed. Two binding parameters – the association constants and number of binding sites on BSA – for each drug were estimated using Scatchard analysis.

Results

Protein binding of chlorpromazine to BSA was reduced with the addition of verapamil whereas that of verapamil was unaffected in the presence of chlorpromazine. Common binding sites on BSA for chlorpromazine and verapamil were observed. Verapamil displayed higher affinity for common binding sites and decreased the association constant of chlorpromazine for these sites.

Conclusion

The displacement of chlorpromazine from common binding sites by verapamil was observed. Such decline in protein binding of chlorpromazine suggested a reduction in the efficacy of chlorpromazine.

Significance and impact of study

Our results suggest that timing of administration of verapamil affects the level of protein binding of chlorpromazine. However, the validity of the finding may be limited which neglects other pharmacokinetic factors and hence may not be clinically relevant. Further investigation should be conducted with careful consideration of other relevant factors.

1. Introduction

1.1 Combination therapy

Every drug has its own indication, its target to act upon and its unique therapeutic profile. In theory, a drug used alone is supposed to treat a disease well. In reality, the effectiveness of a drug can be limited by external factors such as complication, co-morbidity and emergence of resistance. A classic example is the resistance of bacteria with β-lactamase to penicillin. In times when a disease is inadequately controlled by a single drug or when the outcome of monotherapy is unsatisfactory [1], the simultaneous use of two or more drugs, combination therapy in short, can be adopted.

Combination therapy can be beneficial. When one or more agents are added to monotherapy, synergistic effects can result [2]. In a study about antihypertensive treatment, such synergism is observed when the effects of two complementary antihypertensive drugs used in combination are significantly greater than those observed when used individually [3]. This leads to higher efficacy, hence better therapeutic outcome for the treatment of hypertension. Similar conclusion was drawn in the management of acute mania that the efficacy of antipsychotics improves when added to lithium or valproate [1]. Moreover, combination therapy can result in fewer side effects. When side effects are dose-dependent, increasing the dose of the drug in monotherapy can produce more side effects [3]. Therefore, the more desired therapeutic outcome will be attained without jeopardizing patient safety.

While combination therapy can add benefits, it can bring detrimental effects when used inappropriately. In the treatment of hypertension, when the dose of each individual agent is too elevated, the summation of antihypertensive effects can be immense, causing life-threatening problems such as shortness of breath. Hence, combination therapy is always scrutinized and closely monitored to achieve the optimal therapeutic outcome.

Patients are often prescribed complementary drugs which are taken at the same time. The obvious aim is to achieve the desired therapeutic responses. Additionally, it is to enhance compliance rates [3]. However, one should not forget the dynamic interaction between drugs in the body at molecular level. For example, erythromycin inhibits the metabolism of terfenadine by the action of CYP3A4 isozyme, resulting in cardiotoxicity [4]. Verapamil inhibits the P-glycoprotein, hence possibly increasing the penetration of chlorpromazine to the central nervous system [5, 6]. The displacement of one drug from plasma proteins by another drug is another main type of drug-drug interaction. Competitive binding to proteins may alter protein binding and subsequently the pharmacokinetic profile of one drug by another [7-16]. This may in turn accelerate the onset of the therapeutic response of the former drug [7, 8] and may therefore be undesirable. For instance, warfarin was displaced from serum albumin by phenylbutazone [17]. The increase in free warfarin level in the blood could lead to fatal bleeding given that warfarin has a narrow therapeutic index [18]. Hence, the importance of protein binding in drug-drug interactions should be recognized and examined before co-administration of two drugs to avoid any potential adverse effects.

1.2 Protein binding

Protein binding refers to the process of drugs binding to plasma proteins. Most drugs bind to plasma proteins reversibly when they reach systemic circulation [15, 19-21]. Examples of plasma proteins include human serum albumin (HSA), alpha-1 acid glycoprotein (AAG) and lipoproteins. Out of these plasma proteins, HSA and AAG are the main proteins responsible for protein binding [15, 22, 23]. HSA binds primarily to acidic drugs and AAG to basic drugs [15, 24, 25]. Neutral drugs are equally favoured by HSA and AAG [15, 23, 26-28]. Given that the concentrations of HSA are between 3.5-5g/dL, which are significantly more than that of AAG (0.04-0.1g/dL), HSA is the main contributor in protein binding [15].

1.2.1 Concept

A protein molecule has multiple binding sites to which different drugs can bind [7]. Two drugs may bind to different sites or even share a common site. When a drug molecule binds to a protein molecule, a drug-protein complex will be formed. There will be an equilibrium established between the free unbound drug ([D]), the protein ([P]) and the drug-protein complex ([DP]) [15, 29, 30]. This relationship can be illustrated in the following equation:

Equation

where k1 denotes the association constant of DP and k-1 the dissociation constant of DP. Since at equilibrium the rate of association is equal to the rate of dissociation, using these two constants, the association constant Ka and the dissociation constant Kd can be derived. Ka (protein) relates to the affinity of the drug for its binding site on the protein while Kd (protein) is the inverse of Ka (protein) [15]. Below is the equation depicting these relationships:

Equation

Given that a drug has high Ka (protein), [DP] will therefore be high according to equation 2. If another drug that shares the same binding site on a protein as the former drug has an even higher Ka (protein), then the former drug may be displaced, decreasing the bound drug concentration.

The ratio of the free (Cfree) or bound (Cbound) and total (Ctotal) drug concentration can be used to determine the extent of protein binding. This ratio can be expressed as the percentage of drugs bound to protein (%PPB) or the fraction unbound in the protein (fuP):

Equation

Equation

1.2.2 Detection methods

There are several methods used to detect the degree of protein binding. One of the most commonly used methods is equilibrium dialysis.

1.2.2.1 Equilibrium dialysis

Equilibrium dialysis takes place in wells each consisting of two chambers – the sample chamber and the assay chamber – separated by a semi-permeable membrane. The mixture of protein and drug will be added to the sample chamber and the buffer to the assay chamber. The volumes which will be made up in each chamber will be equivalent. The unbound drug will partition from the sample chamber to the assay chamber across the membrane. Since both protein and drug-protein complexes cannot cross the membrane, the assay chamber will only contain free drug. Given that the two chambers are of equal volume, at equilibrium the free drug concentration is equivalent in both chambers. The free drug concentration will be determined by measuring the absorbance of the solution in the assay chamber using a UV-Visible spectrophotometer. The protein-bound drug concentration will be derived by considering the following concept of conservation of mass.

Equation

Equation 5 can be expressed in terms of concentration and volume:

Equation

where C(original) is the original drug concentration added, C(bound) is the bound drug concentration in the sample chamber, C(free) is the free drug concentration in the assay chamber, Vs is the volume in the sample chamber and Va is the volume in the assay chamber.

Since Vs = Va,

Equation

Equation

The degree of protein binding as the bound fraction (fb) can be derived by calculating the unbound fraction (fu), where

Equation

Equation

On the other hand, other binding parameters such as the association constant and number of binding sites on the protein can subsequently be calculated using fb [31].

1.2.3 Importance

Based on the ‘free drug hypothesis’, when a drug is bound by plasma proteins, the concentration of drug available to act on its pharmacological target may be limited [32]. On the contrary, this impact can be reversed by the displacement of a bound drug from a binding site on the protein by another drug. Either way may therefore alter both pharmacokinetic and pharmacodynamic properties of a drug [33, 34]. Since the pharmacological effect of a drug is dependent on these two properties, its clinical therapeutic profile may be changed as a result [7-16, 32].

1.2.3.1 Effects on pharmacokinetics of drugs

Protein binding directly impacts on the pharmacokinetic properties of drugs, mainly volume of distribution and clearance [9-16]. The change in pharmacokinetic profile will subsequently determine the pharmacodynamic properties of drugs. In the following discussion, the two main pharmacokinetic parameters will be examined individually with relation to effects of protein binding.

1.2.3.1.1 Volume of distribution

Volume of distribution (Vd) relates the total amount of a drug in the body to the drug concentration in the body fluid, which is most commonly sampled from plasma [15]. From the following equation, where Vd,plasma is volume of distribution in plasma, Vp is the volume of plasma, fuP and fuT are the fraction of drugs unbound in plasma and tissue respectively and V’T is the tissue volume,

Equation

The direct relationship has been observed between fuP and Vd , ie. the lower the value of fuP , the lower is the value of Vd [15]. For orally administered drugs with high Vd (>30L, eg. amitriptyline) and moderate Vd (3L – 30L, eg. chlorpromazine), the change in fuP will lead to a fully proportional and a less than proportional change in Vd respectively [13, 15, 35, 36]. However, for those with low Vd (<3L, eg. heparin), the change in Vd is very insignificant [15, 36, 37]. As most clinically relevant drugs have moderate Vd, the impact on Vd remains accountable [15].

Since only the unbound drug can diffuse into tissues across membranes and exert its pharmacological effect at the target site, higher levels of protein binding will decrease Vd, subsequently delaying the onset of its pharmacological response and reducing the magnitude of its initial pharmacological response.

1.2.3.1.2 Clearance

Drug-protein complexes act as a reservoir and release the free drug into circulation to maintain the equilibrium [7], or in other words therapeutic response. This equilibrium is disturbed by clearance [15]. As the level of clearance is dependent on Cfree in plasma, the extent of protein binding will hence affect clearance.

Clearance refers to ‘the volume of blood or plasma from which a drug is completely and irreversibly removed per unit time.’ [13, 15] It relates the rate of elimination of a drug to its concentration [38, 39]. The majority of drugs are eliminated by the kidney and/or the liver [15].

For drugs that are extensively metabolized in the liver such as chlorpromazine and verapamil, hepatic clearance could be assumed to account for the entire clearance process.

In hepatic clearance,

Equation

where CLhepatic,plasma is the hepatic clearance of a drug from the plasma; Qhepatic is the liver blood flow; CLuint is the intrinsic clearance based on Cfree; and CB and CP are the drug concentration in blood and in plasma respectively. Assuming CB equals to CP, the equation of CLhepatic,plasma can be rearranged as:

Equation

Hepatic eliminated drugs can be categorized into three groups based on their extraction ratio (E) – low extraction drugs (E<0.3, eg. chlorpromazine), intermediate extraction drugs (E=0.3-0.7, eg. nifedipine) and high extraction drugs (E>0.7, eg. verapamil) [13, 15]. Most drugs are of either low or high extraction types [15, 40]. For low extraction drugs, the product of CLuint and fuP is much lower than Qhepatic, CLhepatic,plasma will be dependent of the extent of protein binding [15, 39, 41]:

Equation

For high extraction drugs, the product of CLuint and fuP is much higher than Qhepatic, CLhepatic,plasma will be insensitive of protein binding [15, 39, 41]:

Equation

In general, protein binding determines the free drug concentration which is disturbed by clearance. The magnitude and the duration of therapeutic effect of a drug will hence be affected.

1.2.3.1.3 Other parameters

Since CL and Vd are factors in determining other parameters, for example average, peak and trough drug concentrations at steady-state as well as half-life, changes in CL and Vd due to the extent of protein binding will manipulate these subsequent parameters [15]. The range of drug concentrations at steady-state reflects the therapeutic profile of a drug. Concentrations higher or lower than the therapeutic concentration will mean toxicity or treatment failure respectively [15]. As half-life is linked to the extent of the pharmacological effect of a drug, any change in half-life may require adjustment in dosing to achieve the desirable therapeutic drug concentration. Due to the complexity in these parameters, the impacts of each parameter due to changes in protein binding are summarized in table 1.

Table . Change in Pharmacokinetic Parameters With Respect to Changes in the Fraction Unbound in Plasma, as summarized by Schmidt, Gonzalez and Derendorf, 2009 [15].

1.2.3.2 Effects on pharmacodynamics of drugs

The process of protein binding is not only confined to plasma but also at the target site of each drug. Proteins are available at the target site, for instance, proteins are present in the cerebrospinal fluid [15, 42]. Hence, free drugs can be bound by these protein [15]. This will limit the amount of free drugs available to elicit the expected level of therapeutic effect [15]. For drugs which have target sites in the central nervous system (CNS) such as chlorpromazine, their efficacy may hence be reduced.

1.3 Chlorpromazine

Chlorpromazine is a first-generation antipsychotic drug developed and it is a Group 1 phenothiazine derivative. It is available in two salt formulations - chlorpromazine embonate and chlorpromazine hydrochloride - to improve its solubility [43]. Of these two salts, the latter is more widely used.

1.3.1 Chemical structure and properties

As seen in figure 1, chlorpromazine has a basic cationic nature owing to the tertiary amine present which has two methyl groups attached. This amine can be easily protonated. As a hydrochloride salt, chlorpromazine is soluble 1 in 2.5 of water which gives a decent dissolution profile. It has a molecular weight of 355.3 [43]. Its pKa is 9.3 at 20ËšC [43], giving it a weakly basic feature. It is lipophilic with Log P (octanol pH 7.4) being 3.4; however, its amphipathic nature has also been noted [43, 44].

Figure

Figure . Chemical structure of chlorpromazine [43].

1.3.2 Pharmacokinetics

Chlorpromazine has the following pharmacokinetic profile:

Absorption: Chlorpromazine is well absorbed from the gut following oral administration and its plasma concentration peaks after 2-4 hours [43].

Distribution: It is widely distributed in the body with a volume of distribution of about 21 L/kg [43, 45] and across the blood brain barrier, attaining a higher concentration in the brain than in plasma [46]. It is about 95-98% plasma proteins bound, mostly to serum albumin. It also binds to two major blood components – membranes of erythrocytes and lipoproteins [47-53]. In addition, it is observed to be extensively localized in tissues [54].

Metabolism: Chlorpromazine undergoes extensive first-pass metabolism in the liver, catalysed by the P450 isoenzyme CYP2D6. Some paths of metabolism include hydroxylation, glucuronic acid conjugation, sulfoxidation, N-demethylation, and N-oxidation [46]. This results in a low bioavailability of approximately 20-30% [43]. Two of its active metabolites isolated are 7-hydrochlorpromazine and nor1-chlorpromazine [43, 55].

Excretion: About 20-70% of oral doses of chlorpromazine are excreted in the urine, mostly as conjugated metabolites with clearance from plasma of around 8.6 mL/min/kg. Its mean half-life of elimination is in the range of 15-30 hours [43].

In addition, the effective therapeutic plasma concentration of chlorpromazine has been observed to be in the range of 0.03-0.3 mg/L. Above which, toxicity has been observed [56].

1.3.3 Pharmacodynamics

Chlorpromazine has been observed to show affinities to dopamine D1 and D2 receptors [2]. However, it acts predominantly by blocking the postsynaptic dopamine D2 receptors in the CNS, particularly in the mesolimbic-frontal system [2]. This antagonising action suppresses the excessive dopaminergic activity, thereby achieving the antipsychotic effect [2].

It has been observed that chlorpromazine is a substrate to P-glycoprotein (P-gp) [6, 57]. P-gp is present in the apical membranes of epithelial cells found in the body such as the enterocytes in the intestine and the brain capillary endothelial cells that constitute the blood-brain barrier [58, 59]. Since P-gp is responsible for drug efflux, P-gp will limit the brain penetration of chlorpromazine [58].

1.3.4 Uses

The main use of chlorpromazine is to treat schizophrenia and other psychoses such as mania. Other uses include the treatment of nausea and intractable hiccup [60]. It is administered orally, intramuscularly and rectally for these indications [60].

On the other hand, chlorpromazine has been used clinically in combination with other drugs to treat psychoses [61]. One example of these drugs is verapamil.

1.4 Verapamil

Verapamil is a calcium-channel blocker, widely used for cardiac problems such as hypertension. It is formulated as verapamil hydrochloride [43].

1.4.1 Chemical structure and properties

As seen in figure 2, verapamil has a basic cationic nature owing to the tertiary amine present. This amine can be easily protonated. As a hydrochloride salt, verapamil is soluble 1 in 20 of water. It has a molecular weight of 491.1 [43]. Its pKa is 8.9 and it is lipophilic with Log P (octanol/water) being 3.8 [43].

Figure

Figure . Chemical structure of verapamil [43].

1.4.2 Pharmacokinetics

Verapamil has the following pharmacokinetic profile:

Absorption: Over 90% on average of verapamil is absorbed after oral administration [43, 62-64]. Its plasma concentration peaks within 1-2 hours after an oral dose [64-66].

Distribution: Its apparent volume of distribution is approximately 2.5L/kg [64]. Over 90% of verapamil is bound to proteins [62, 64]. Low binding of verapamil to red blood cells was reported [64, 67].

Metabolism: Verapamil undergoes extensive first-pass metabolism [43, 64]. This results in a low bioavailability of about 20% [2]. Verapamil is primarily metabolized by N-dealkylation and O-demethylation into several metabolites of which the only active metabolite produced is norverapamil [43, 64].

Excretion: About 70% of a dose is renally excreted in 5 days with approximately 5% excreted unchanged. N-dealkylated compounds are the main urinary metabolites with about 10% of the dose as norverapamil. Approximately 17% of the urinary materials are made up with O-demethylated derivatives. About 16% of a dose is excreted in the faeces [43]. The elimination half-life of verapamil is around 4-5 hours [64, 68].

In addition, the effective therapeutic plasma concentration of verapamil has been observed to be in the range of 15-100 mg/L [69].

1.4.3 Pharmacodynamics

Verapamil inhibits the influx of calcium ions through the voltage-dependent L-type calcium ion channels present in myocardial and vascular smooth muscle cell membranes [2, 60] as well as the nerve cell membranes in the CNS [61]. This inhibition suppresses the formation and propagation of electrical impulses, thereby reducing the contractile response of the cardiac muscles [60].

It has been shown that verapamil is a competitive inhibitor of P-gp [5, 6]. Hence, verapamil can enhance brain penetration of substrates of P-gp such as chlorpromazine.

1.4.4 Use as an adjunct

In addition to its primary use in treating angina, hypertension and arrhythmias, verapamil can be used to treat mania [61, 70-75]. It was reported that intracellular calcium level is elevated in mania [75, 76]. Blocking the influx of calcium into neurons may reduce the conductivity of neurons in the CNS, possibly relieving the manic condition [75]. Despite that, for the treatment of severe mania, verapamil when used alone shows no comparable effectiveness to agents such as lithium and chlorpromazine [61, 75]. On the contrary, when verapamil is used as an adjunct to these better agents, clinically useful additive or synergistic effects have been observed [61, 75]. Therefore, verapamil has been used in combination with the better agents when manic patients fail to respond to these agents alone.

1.5 Justification

In previous sections, we have discussed the impacts of protein binding and the potential effectiveness of combination therapy. We also looked into the pharmacokinetic and pharmacodynamic profiles of chlorpromazine and verapamil. Given that both drugs have high levels of protein binding, the possible dynamic interaction would be of interest as to whether the efficacy of chlorpromazine will be affected.

In the remaining sections of this report, we aim to investigate the protein binding chemistry of these two drugs when they are administered together for the treatment of mania. The binding of drugs to bovine serum albumin (BSA) will be studied because of similar structural homology of BSA with HSA as well as its low cost [77-81]. The findings could possibly provide useful information with regards to the use of combination therapy, thereby enhancing the therapeutic outcome.

3. Materials and methods

3.1 Reagents

Chlorpromazine hydrochloride (CPZ), verapamil hydrochloride (VPL) and bovine serum albumin (BSA) (≥98%, A7906) were all purchased from Sigma-Aldrich Co. UK. The molecular weight of BSA was approximately 66,430 [82]. Universal buffer of pH7.4, containing 10mM Citric acid monohydrate, 10mM Sodium dihydrogen orthophosphate and 10mM Boric acid, was used. All other materials including 98% ethanol and distilled water were of analytical grade.

3.2 Apparatus

UV-Visible absorption spectra of solutions were produced using the Cary 4000 UV-Visible Spectrophotometer and the Cary WinUV software both purchased from Varian, Inc., UK. Synthetic quartz glass cuvettes with 1cm path length were used for the measurement.

UV absorbance of solutions was measured using quartz cuvettes with 1cm path length and the Cecil spectrophotometer (CE-1021, Cecil Instruments, UK).

Equilibrium dialysis was carried out on a 96-well Micro-Equilibrium Dialysis Device (HTD96b) with dialysis membrane strips (MWCO: 12-14kDa) and adhesive sealing films. These were purchased from HTDialysis LLC, UK.

List of common apparatus used was detailed in appendix 3.

3.3 Calibration curves

Calibration curves are used to determine an unknown concentration of a solution based on the absorbance reading at a particular wavelength. Before a calibration curve can be created, this particular wavelength must be ascertained. Therefore, a solution of a particular drug would be made up and scanned using the Cary 4000 UV-Visible Spectrophotometer and the Cary WinUV software to obtain an UV-Visible absorption spectrum. The wavelength at which optimal absorption takes place would be identified. Subsequent solutions of this particular drug would be measured at this wavelength to derive respective concentrations.

3.3.1 UV-Visible absorption spectra

3.3.1.1 Chlorpromazine hydrochloride

10mg of CPZ was weighed out and placed into a vial. Because of its low solubility in universal buffer, instead of producing a 10mL solution, CPZ was dissolved using universal buffer to make into a 100mL solution in a 100mL volumetric flask. 10mL of universal buffer was added to the vial and the vial was shaken. The content was subsequently transferred to the flask. The vial was rinsed out with universal buffer properly several times to ensure complete transfer of CPZ into the flask. The solution in the flask was carefully made up to 100mL to produce a 0.1mg/mL solution. The flask was shaken thoroughly to ensure complete dissolving.

A quartz glass cuvette was filled with universal buffer. This cuvette was inserted into the holder in the Cary 4000 UV-Visible Spectrophotometer and was scanned. This was to detect any background absorbance reading. Using the option ‘Baseline correction’, the background absorbance would be removed from the absorbance of subsequent samples. Another cuvette filled with 0.1mg/mL CPZ solution was inserted and scanned to produce the UV-Visible absorption spectrum of CPZ (Figure 3). In this spectrum, CPZ peaked at 306nm. At this wavelength, further absorbance measurements of CPZ solutions would be performed.

FIGURE .UV-Visible absorption spectrum of CPZ.

3.3.1.2 Verapamil hydrochloride

10mg of VPL was used in the same procedure in section 3.3.1.1. Figure 4 showed the UV-Visible absorption spectrum of VPL in which VPL peaked at 230nm and 278nm.

Figure . UV-Visible absorption spectrum of VPL.

Comparing both UV-Visible absorption spectra of CPZ and VPL in figure 5, the spectrum of VPL entirely overlapped with that of CPZ.

Figure .UV-Visible absorption spectra of CPZ and VPL.

In a mixture of CPZ and VPL, the absorbance of VPL would not be accurately distinguished from that of CPZ whereas the absorbance of CPZ could be determined at 306nm at which VPL exhibited zero absorbance. To determine the concentration of VPL in a mixture of CPZ and VPL, one solution would be to measure the mixture at a wavelength at which the absorbance of an individual drug could confidently and reliably be deduced. Given that absorbance of CPZ could be reliably measured at 278nm and the concentration of CPZ would be derived at 306nm, a calibration curve for CPZ at 278nm would be useful to derive the absorbance of CPZ at 278nm using the concentration of CPZ obtained at 306nm. The process of derivation of absorbance of CPZ was depicted in equations 16-18.

Equation

Equation

Equation

As a result, the concentration of VPL at 278nm could be obtained by subtracting the absorbance of CPZ at 278nm from the total absorbance of the mixture at 278nm, as shown in equations 19-20.

Equation

Equation

Hence, a total of three calibration curves would be produced to determine respective concentrations of CPZ and VPL: two calibration curves for CPZ at 278nm and 306nm and one calibration curve for VPL at 278nm.

3.3.2 Preparation of calibration curves

3.3.2.1 Chlorpromazine hydrochloride

Following the same procedure stated in section 3.3.1.1 in preparing CPZ solution, 10mg of CPZ was dissolved in universal buffer to produce 100mL of 0.1mg/mL CPZ solution. Approximately 10mL of this solution was poured from the flask to a labeled vial. This CPZ solution would be measured for absorbance at 278nm and 306nm.

The Cecil spectrophotometer was switched on and the wavelength was set as 278nm. Approximately 2mL of universal buffer was added to a quartz cuvette which was placed in the holder. The Cecil spectrophotometer was tared to remove any absorbance reading contributed by the universal buffer. Approximately 2mL of 0.1mg/mL CPZ solution was added from the labeled vial to a cuvette and was measured. The absorbance was recorded and the same procedure was repeated at 306nm.

At wavelengths of 278nm and 306nm, the absorbance readings were 0.562 and 1.088 respectively which are both below the value of 1.1. Any absorbance values above this are considered to be unreliable because of a non-linear increase in absorbance against concentration. Hence, 0.1mg/mL CPZ solution was employed to be the starting solution for the dilution series. The plan to produce a serial dilution was detailed in appendix 3.

Four solutions at CPZ concentrations (0.01mg/mL, 0.02mg/mL, 0.04mg/mL and 0.08mg/mL) were made up in four different vials which were labeled respectively. The vials were shaken thoroughly to ensure complete mixing. Absorbance of these four solutions were subsequently measured and recorded at 278nm and 306nm following the above-mentioned procedure. The entire procedure was repeated twice to improve the reliability of results in preparing calibration curves.

Results from three repeats were tabulated using Microsoft Excel software. The concentration was modified to be expressed in molarity for convenient calculation in subsequent experiments. Two calibration curves for CPZ at 278nm and 306nm were plotted with two regression equations derived at each wavelength.

3.3.2.2 Verapamil hydrochloride

Following the same procedure stated in section 3.3.2.1, 10mg of VPL was used instead to produce 100mL of 0.1mg/mL VPL solution.

The Cecil spectrophotometer was tared against universal buffer at 278nm. The 0.1mg/mL VPL solution was measured for its absorbance. Given that the recorded absorbance of this solution was 0.956, this solution was used as a starting solution in the dilution series for VPL in preparing the calibration curve for VPL. The plan for serial dilution for VPL was detailed in appendix 3.

Four solutions with different VPL concentrations (0.00625mg/mL, 0.0125mg/mL, 0.025mg/mL and 0.05mg/mL) were made up in four labeled vials. These vials were shaken thoroughly to ensure complete mixing. Absorbance of these four solutions were subsequently measured and recorded at 278nm. Two repeats of this experiment were performed. Results from three repeats were tabulated using Microsoft Excel software. The concentration was modified to be expressed in molarity. As a result, a calibration curve for VPL with a regression equation was obtained.

3.4 Equilibrium dialysis

Individual and combined studies of CPZ and VPL on their binding to BSA were performed using equilibrium dialysis. Such experiments were designed to observe the changes in free drug concentrations against the changes in original drug concentrations, thereby investigating the degree of protein binding. In addition, the results of protein binding were analysed using Scatchard plots to estimate the association constants and number of binding sites on BSA for both drugs.

3.4.1 Preparation of stock solutions

3.4.1.1 Bovine serum albumin

66.4mg of BSA was accurately weighed out and placed into a vial. 10mL of universal buffer was pipetted to the vial to produce a 0.1mM solution. This solution was properly mixed in the Stirling mixer. The whole procedure was performed carefully and gently to avoid any foaming in the solution. The solution was stored in the refrigerator until use.

3.4.1.2 Chlorpromazine hydrochloride

17.8mg of CPZ was accurately weighed out and placed into a vial. CPZ was dissolved using universal buffer to make into a 100mL solution in a 100mL volumetric flask. 10mL of universal buffer was added to the vial and the vial was shaken. The content was subsequently transferred to the flask using a funnel. The vial was rinsed out with universal buffer properly several times into the flask. The solution was carefully made up to 100mL to produce a 0.5mM solution. The flask was shaken thoroughly to ensure complete dissolving.

3.4.1.3 Verapamil hydrochloride

The procedure in section 3.4.1.2 was repeated using 24.6mg of VPL to produce 100mL of 0.5mM VPL solution.

3.4.2 Experimental design

3.4.2.1 Individual studies on the binding to bovine serum albumin

3.4.2.1.1 Chlorpromazine hydrochloride

300µL of 0.1mM BSA stock solution was each added to five vials. According to table 2, different volumes of 0.5mM CPZ stock solution and universal buffer were added to these vials to produce five solutions containing BSA and CPZ with different CPZ concentrations (100µM, 200µM, 250µM, 300µM and 400µM). These vials were labeled respectively, mixed properly using the Stirling mixer and allowed to stand for 30 minutes for maximum binding of CPZ to BSA.

Table . Proportions of various solutions in constituting five solutions with CPZ concentrations 100µM, 200µM, 250µM, 300µM and 400µM.

In table 2, the concentrations of BSA in all solutions were fixed. This was to observe how free CPZ concentrations change at different concentrations of CPZ. On the other hand, the ratios of concentrations of BSA to concentrations of CPZ were set to be lower than the physiologically observed values. The reason was that due to high protein binding of CPZ, free concentrations of CPZ would be too low to be detected from one well. Therefore, the ratios of concentrations of BSA to concentrations of CPZ were amended and dialysate from each of eight wells would be combined to make a single measurement. This would ensure free CPZ concentrations to be measurable. The volume of each solution was made up to sufficiently fill the sample chambers of eight wells.

The 96-well Micro-Equilibrium Dialysis Device was properly assembled with dialysis membrane strips inserted. The wells were filled with universal buffer to keep membrane strips hydrated until use.

The wells were categorized using labels in tables 3 and 4. In each well, 150µL of a solution containing BSA and CPZ was added to the sample chamber using a 20-200µL micropipette whereas 150µL of universal buffer was added to the assay chamber. Two controls were added. Control 1 was to ensure that no protein crossed the membrane. Control 2 was to check for the equilibrium time at which both chambers exhibited equivalent absorbance. This was done by diluting 100µL from each chamber with 2mL of universal buffer and measuring free drug concentrations. The sealing film was applied to allow incubation. The Micro-Equilibrium Dialysis Device was placed in the electronic shaker until equilibrium was reached.

Table . Individual labels assigned to each test and control.

Table . Diagrammatic representation of the plan for equilibrium dialysis.

When equilibrium was reached, 100µL dialysate from each of the eight wells was extracted from the assay chamber and diluted with 1200µL of universal buffer. Free concentrations of CPZ of these diluted dialysates were measured by the Cecil spectrophotometer at 306nm. The results were recorded and this experiment was repeated twice.

3.4.2.1.2 Verapamil hydrochloride

The same procedure in section 3.4.2.1.1 was carried out using 0.5mM VPL stock solution instead of CPZ stock solution. The proportions to constitute five solutions containing BSA and VPL with different VPL concentrations (100µM, 200µM, 250µM, 300µM and 400µM) were shown in table 5. Free concentrations of VPL of the diluted dialysates were measured by the Cecil spectrophotometer at 278nm. The results were recorded and this experiment was repeated twice.

Table . Proportions of various solutions in constituting five solutions with VPL concentrations 100µM, 200µM, 250µM, 300µM and 400µM.

3.4.2.2 Displacement studies on the binding to bovine serum albumin

These studies were to investigate mutual effect of CPZ and VPL on the binding to BSA based on the changes in respective free drug concentrations at different drug concentrations. These studies also assessed whether the order of addition of CPZ and VPL to BSA would have an effect on protein binding of the respective drugs.

3.4.2.2.1 Effect of verapamil hydrochloride on chlorpromazine hydrochloride

Following similar procedures stated in section 3.4.2.1.1, six identical solutions containing BSA and CPZ (molar ratio of BSA:CPZ, 20µM: 200µM, 1: 10) were constituted in six labeled vials using BSA and CPZ stock solutions according to table 6. These vials were mixed properly using the Stirling mixer and were allowed to stand for 30 minutes for maximum binding of CPZ to BSA.

Table . Proportions of various solutions used in constituting six solutions of BSA, CPZ and VPL.

To each of these six solutions containing BSA and CPZ, different volumes of VPL stock solution and universal buffer, stated in table 6, were added to the respective vials to make six different solutions containing BSA, CPZ and VPL. The total volume in each vial was sufficient to fill eight wells. These vials were again mixed properly using the Stirling mixer and allowed to stand for 30 minutes.

The 96-well Micro-Equilibrium Dialysis Device was properly assembled and prepared. The wells were categorized using labels in tables 7 and 8. In each well, 150µL of a solution containing BSA, CPZ and VPL was added to the sample chamber whereas 150µL of universal buffer was added to the assay chamber. Three controls were added. Control 1 was to ensure no protein crossed the membrane. Controls 2 and 3 were to check for the equilibrium time for each drug. The sealing film was applied. The Micro-Equilibrium Dialysis Device was placed in the electronic shaker for equilibration.

Table . Individual labels assigned to each test and control.

Table . Diagrammatic representation of the plan for equilibrium dialysis.

When equilibrium has been reached, 100µL dialysate from each of the eight wells was extracted from the assay chamber and diluted with 1200µL of universal buffer. Free concentrations of CPZ of these diluted dialysates were measured by the Cecil spectrophotometer at 306nm whereas free concentrations of VPL were measured at 278nm based on equation 20. The results were recorded and this experiment was repeated twice.

3.4.2.2.2 Effect of chlorpromazine hydrochloride on verapamil hydrochloride

The same procedure in section 3.4.2.2.1 was carried out, rotating the position of CPZ and VPL. Six solutions containing BSA and VPL (molar ratio of BSA:VPL, 20µM: 200µM, 1:10) were made up initially. Different volumes of CPZ stock solution and universal buffer were added subsequently to create six solutions containing BSA, VPL and CPZ, with reference to table 9.

Table . Proportions of various solutions used in constituting six solutions of BSA, CPZ and VPL.

At the end of this experiment, free concentrations of CPZ of the diluted dialysates were measured by the Cecil spectrophotometer at 306nm whereas free concentrations of VPL were measured at 278nm based on equation 20. The results were recorded and this experiment was repeated twice.

3.4.2.3 Calculation of the percentage of protein binding

The results of free drug concentrations were used to calculate the percentages of protein binding (%PPB). Based on equation 3, %PPB is given by:

Equation

where Doriginal represents the original drug concentration in the sample chamber; Dbound is the bound drug concentration in the sample chamber and Dfree is the free drug concentration in the assay chamber.

3.4.2.4 Estimation of association constants and number of binding sites on bovine serum albumin

The values of free drug concentrations obtained were used to produce Scatchard plots to estimate the association constants and number of binding sites on BSA for both CPZ and VPL [83, 84]. In such Scatchard plots, r/Dfree was plotted against r where r depicts the ratio of the Dbound to the total BSA concentration (Ptotal), as shown in the following equation [85]:

Equation

These plots were extrapolated to produce straight lines. The intersection between straight lines and the Y-axis yields the nKa values and the slope of the lines gives -Ka, where n is the number of binding sites of a particular drug on BSA and Ka is the association constant of that drug to BSA.

3.5 Statistical analysis

Statistical analysis was performed on all results using Microsoft Excel software. Mean and standard deviation were calculated for the results within each experiment. Experimental results for each experiment were expressed as mean± standard deviation. One-way ANOVA test and unpaired T-Test were performed where appropriate.

4. Results and discussion

4.1 Calibration curves

A total of three calibration curves were obtained: two calibration curves for CPZ at wavelengths of 278nm and 306nm and one for VPL at a wavelength of 278nm. They were plotted in a single graph as shown in figure 6. Each calibration curve was accompanied by a regression equation which was used to determine the corresponding drug concentration from the observed absorbance.

Figure 6. Calibration curves for CPZ and VPL at different wavelengths (278nm and 306nm).

All three curves showed good linearity with coefficients of determination (R2) close to 1: R2 = 0.9967 for CPZ at 278nm, R2 = 0.9984 for CPZ at 306nm and R2 = 0.998 for VPL at 278nm. These regression lines fit their respective data well and hence provided a confident and reliable measure of corresponding drug concentrations. However, these calibration curves were plotted based on the absorbance readings obtained at different concentrations but not at identical concentrations. One limitation was that the range of absorbance readings at identical concentrations, represented using y-error bars, was not recognized. Significant deviations in absorbance readings might be neglected, thereby potentially reducing the accuracy and reliability in deducing corresponding drug concentrations. It is therefore recommended in future work to measure absorbance values at identical concentrations in the preparation of calibration curves.

As discussed previously, in a mixture of CPZ and VPL, the absorbance of VPL was deduced by subtracting the absorbance of CPZ from the total absorbance at 278nm. Significant errors might arise due to this indirect derivation, thereby running the risk of underestimating or overestimating the concentration of VPL present. Therefore, the experiment in preparing calibration curves for CPZ was repeated five times to improve the reliability of experimental data. Moreover, investigation was performed to confirm that the total absorbance of a mixture of CPZ and VPL was the sum of the individual absorbance (Refer to appendix 4). It was shown that absorbance was additive. It also validated the use of individual regression equations that percentages of errors in deriving the corresponding concentrations of CPZ and VPL were less than 5%. Furthermore, based on equations 18-20, the regression equation for VPL had been modified for convenient calculation which is shown in table 10.

Table . Regression equations of calibration curves for CPZ and VPL where y represents the absorbance value, xc represents the concentration of CPZ and xv represents the concentration of VPL.

Regression equation

CPZ at 306nm

y = 3.7015xc

VPL at 278nm

y = 4.766xv + 1.9426xc

4.2 Equilibrium dialysis

4.2.1 Determining the percentage of protein binding

Prior to analyzing the experimental data, results from several controls were examined to ensure that the system was behaving as expected and to allow one to identify any sources of error in need of adjustment. Both sample chambers and assay chambers in all controls were sampled and measured for absorbance. All results showed that the absorbance fell within the reference absorbance inclusive of a 5% error (Refer to appendix 4). This demonstrates that no protein crossed the membrane and no drugs adsorbed to the dialysis device or membrane. These control data indicated that no or minimal source of error was present in the system used for the experiments.

Experimental data in both individual studies and displacement studies were analyzed. Respective free drug concentrations were used to calculate the percentage of protein binding of CPZ and VPL.

Figure 7. Protein binding of CPZ and VPL to BSA in individual studies of the binding of drugs to BSA. Total concentration of BSA = 20µM. Total concentrations of CPZ = 100µM, 200µM, 250µM, 300µM and 400µM. Total concentrations of VPL = 100µM, 200µM, 250µM, 300µM and 400µM. Values represent three consecutive experiments and were expressed as mean± standard deviation. One-way ANOVA test was performed at data points of individual protein binding at five respective concentrations. P-value for protein binding of CPZ at five concentrations was 1.1 x 10-5 whereas p-value for protein binding of VPL at five concentrations was 2.7 x 10-5.

Figure 7 shows individual protein binding of CPZ and VPL at different respective concentrations. It was observed that protein binding of both CPZ and VPL decreased gradually with increasing concentration of the respective drug. Protein binding of CPZ dropped from 63% ± 4% at 100µM concentration to 41% ± 1% at 400µM (P<0.0001) whereas protein binding of VPL dropped from 50% ± 6% at 100µM to 25% ± 1% at 400µM (P<0.0001). All points were accompanied by low standard deviations except the points at 100µM CPZ and VPL concentrations. This was due to lower free drug concentrations present, thereby causing higher level of fluctuations of absorbance values. Combining more wells for testing could improve the coherence of the absorbances.

The decline observed in figure 7 was a result of saturable concentration-dependent binding. The p-values of the two individual plots agreed that individual protein binding of CPZ and VPL was concentration-dependent. This type of binding occurs at high drug concentrations induced by rapid intravenous administration of drugs and an increase in the frequency of therapeutic dosing [28]. In such binding, the binding sites on the protein were saturated with drug molecules. This results in a higher fraction of drug unbound, hence a lower level of protein binding. In figure 7, it was observed that levels of individual protein binding of both drugs were not in agreement with the reference protein binding stated in the literature. This is due to the fact that the ratio of BSA concentration to drug concentration was several folds lower than the ratio at physiological and therapeutic concentrations.

Figure 8. Protein binding of CPZ and VPL to BSA in the displacement study of the effect of VPL on the binding of CPZ to BSA. VPL was added to the mixture of CPZ and BSA (molar ratio of BSA:CPZ, 20µM:200µM, 1:10). Total concentration of BSA = 20µM. Total concentration of CPZ = 200µM. Total concentration of VPL = 100µM, 120µM, 150µM, 180µM and 200µM. Values represent three consecutive experiments and were expressed as mean± standard deviation. One-way ANOVA test was performed at all data points of protein binding of CPZ and at all data points of protein binding of VPL. P-value for protein binding of CPZ was 3.0 x 10-6 whereas p-value for protein binding of VPL was 1.4 x 10-9.

A decline in protein binding for both CPZ and VPL was also seen in figure 8 where VPL was added to the mixture of CPZ and BSA (molar ratio of BSA:CPZ, 20µM:200µM, 1:10). This displacement study was to simulate a scenario in which VPL was orally administered after the oral administration of CPZ on separate occasions. Any effect of VPL on the binding of CPZ to BSA was examined. It was observed that CPZ experienced approximately 25% reduction in protein binding after 200µM VPL was introduced (P<0.00001). Protein binding of VPL declined from 49%±3% at 100µM to 35%± 3% at 200µM (P<0.00000001). Such a decline exhibits a high degree of similarity to protein binding of VPL in the individual study at corresponding concentrations in figure 7. Binding of VPL to BSA was not interfered by the presence of CPZ. The decline in protein binding of VPL was a result of saturable concentration-dependent binding. In contrast, the decline in protein binding of CPZ was caused by the action of displacement by VPL, since concentrations of CPZ used were maintained at 200µM. The displacement of CPZ by VPL suggests that VPL competes with CPZ at the same binding site and VPL has a higher association constant for the common binding sites on BSA than CPZ.

Figure 9. Protein binding of CPZ and VPL to BSA in the displacement study of the effect of CPZ on VPL binding to BSA. CPZ was added to the mixture of VPL and BSA (molar ratio of BSA:VPL, 20µM:200µM, 1:10). Total concentration of BSA = 20µM. Total concentration of VPL = 200µM. Total concentration of CPZ = 100µM, 120µM, 150µM, 180µM and 200µM. Values represent three consecutive experiments and were expressed as mean± standard deviation. One-way ANOVA test was performed at all data points of protein binding of CPZ and at all data points of protein binding of VPL. P-value for protein binding of CPZ was 1.3 x 10-6 whereas p-value for protein binding of VPL was 1.3 x 10-6.

Figure 9 shows the level of protein binding of CPZ and VPL in another displacement study where CPZ was added to the mixture of VPL and BSA (molar ratio of BSA:VPL, 20µM:200µM, 1:10). Protein binding of VPL appeared to be relatively constant and unaffected by the presence of CPZ. Its protein binding was maintained around 35% (P<0.00001). In contrast, protein binding of CPZ experienced slight decline from 48% ± 9% at 100µM to 39% ± 2% at 200µM (P<0.00001), which was lower than its individual binding to BSA at the corresponding concentrations in figure 7. Such results showed that VPL was not displaced by CPZ from the common binding sites on BSA and CPZ was bound to BSA to a smaller extent. This suggests that CPZ might bind to other binding sites that VPL was excluded from or that VPL has weaker affinity for. Even though the slight decline in the level of protein binding of CPZ was shown to be significant as seen from the associated p-value, the overlap of the error bars at each concentration of CPZ might not support this statement. One solution would be to compare protein binding of CPZ at 200µM across three different studies, since protein binding of CPZ at 200µM was investigated in three studies. This comparison is displayed in figure 10.

Figure 10. Comparison of individual protein binding of CPZ and VPL to BSA in different studies. Across three studies, BSA concentration = 20µM, CPZ concentration = 200µM and VPL concentration = 200µM. Values were expressed as mean± standard deviation. Unpaired T-Test was performed for protein binding of respective drugs in two displacement studies. P-value for protein binding of CPZ was 0.027 whereas p-value for protein binding of VPL was 0.53.

In figure 10, protein binding of both CPZ and VPL at 200µM was compared across three different studies. Protein binding of CPZ was confidently seen to have decreased in the presence of VPL; and it reduced more when VPL was bound to BSA first (P< 0.03). The p-value indicated that the order of addition of VPL to CPZ played a significant role in determining the level of protein binding. On the contrary, protein binding of VPL remained relatively constant (P>0.5). The associated p-value indicated that there was no observable difference in the level of protein binding of VPL regardless of the order of addition of CPZ to VPL. The rationale behind these phenomena should be further discussed at the molecular level by looking at the number of binding sites and affinity for these binding sites for each drug. This investigation might be useful in interpreting and explaining the incoherent observation of protein binding of CPZ and VPL.

4.2.2 Determining the association constant and number of binding sites on bovine serum albumin

Two binding parameters – the association constant (Ka) and number of binding sites on BSA (n) – of CPZ and VPL have been characterized using Scatchard plots. Figures 11 and 12 show the individual Scatchard plot for each drug.

Figure . The Scatchard plot of CPZ binding to BSA in two studies: individual study of CPZ alone and displacement study of effect of CPZ on the binding of VPL to BSA.

Figure 12. The Scatchard plot of VPL binding to BSA in two studies: individual study of VPL alone and displacement study of effect of VPL on the binding of CPZ to BSA.

In both figures 11 and 12, non-linear curves had been observed, indicating the presence of two classes of binding sites on BSA for each drug [85, 86]. The two classes of binding sites are class l (high affinity, low capacity) and class ll (low affinity, high capacity). Individual values of Ka and n for both classes of binding sites had been calculated and summarized in table 11.

Table . The association constants and number of binding sites of CPZ and VPL for the binding to BSA. aExperimental data collected from the displacement study of the effect of CPZ on VPL binding to BSA. bExperimental data collected from the displacement study of the effect of VPL on CPZ binding to BSA.

CPZ

VPL

Ka (x105 M-1)

n

Ka (x105 M-1)

n

Class l

Drug alone

0.24

5.94

0.54

2.61

Drug + another drug

0.21a

3.73a

0.66b

2.34b

Class ll

Drug alone

0.09

9.23

0.07

5.08

Drug + another drug

0.09a

6.09a

-

-

In table 11, it was calculated that there were 5.94 class l binding sites on BSA for CPZ alone and the association constant for these binding sites was 0.24 x 105 M-1. 9.23 class ll binding sites were discovered for CPZ alone and the association constant was 0.09 x 105 M-1. After VPL was bound to BSA, only 3.73 class l binding sites and 6.09 class ll binding sites for CPZ remained. Binding sites of both classes reduced their availability to binding of CPZ with a loss of 2.21 class l binding sites and a loss of 3.14 class ll binding sites for CPZ. Despite the loss of binding sites, the association constants for class ll binding sites stayed at approximately 0.09 x 105 M-1 but dropped slightly for class ll binding sites from 0.24 x 105 M-1 to 0.21 x 105 M-1.

In comparison, for VPL alone, there were 2.61 class l binding sites on BSA and the association constant was 0.54 x 105 M-1 while there were 5.08 class ll binding sites on BSA and the association constant was 0.07 x 105 M-1. After CPZ was bound to BSA, the number of class l binding sites on BSA for VPL was 2.34 and the association constant was 0.66 x 105 M-1. Number of class ll binding sites could not confidently be determined as there were insufficient points in the Scatchard plot. Class l binding sites for VPL recorded an insignificant loss of 0.27 binding sites while the association constant for class l binding site increased from 0.54 x 105 M-1 to 0.66x 105 M-1.

The above-mentioned results suggest that class l binding sites for VPL are a subset of that for CPZ. There are approximately 2.34 common class l binding sites shared between CPZ and VPL whereas about 3.73 class l binding sites are solely for the binding of CPZ. VPL displayed higher affinity for the common class l binding sites than CPZ based on the respective values of the association constant. This well accounts for the reduction in protein binding of CPZ because CPZ was displaced from these shared class l binding sites by VPL. However, there is insufficient evidence to account for the difference in protein binding of CPZ when VPL was introduced before and after the administration of CPZ. Despite this, it can be concluded that CPZ and VPL underwent competitive binding to BSA and VPL was a displacer to CPZ.

4.2.3 Implications

As discussed previously, changes in protein binding of a drug may result in changes in its pharmacokinetic properties. This may in turn lead to a change in pharmacological effect of a drug [7, 8]. Therefore, two primary pharmacokinetic parameters – volume of distribution and clearance - of a drug have to be examined with regards to the changes in protein binding. The resultant pharmacokinetic profile will hence determine whether changes in protein binding are of any clinically relevance. Since protein binding of VPL remained relatively constant, only the effects of protein binding of CPZ will be focused.

Given that CPZ has a moderate volume of distribution (21L) [43, 45, 87], based on equation 11, an increase in fraction unbound of CPZ will result in a less than proportional increase in volume of distribution. The distribution of CPZ into tissues will be expected to increase slightly. On the other hand, CPZ is a low extraction drug [88]. Based on equation 14, an increase in fraction unbound will lead to a rise in hepatic clearance which will in turn increase the magnitude of total clearance. Since the increase in total clearance will be more than the increase in volume of distribution, according to table 1, half-life and the free peak concentration of CPZ at steady-state will decrease slightly. This indicates that, when used with VPL, the initial pharmacological effect of CPZ will be slightly lower and its entire duration will be shorter than when CPZ is used alone. Therefore, combination therapy of CPZ and VPL may slightly reduce the efficacy of CPZ as a result of direct displacement of CPZ by VPL.

The reduction in the efficacy of CPZ may be expected more when CPZ was administered before VPL. This further suggests that oral administration of VPL should be prior to that of CPZ in order to achieve better clinical outcome. Even though the reduction in efficacy of CPZ may be marginal, adjustment in dosing may be considered when these two drugs are to be administered together.

4.2.4 Limitations and possible improvements in further studies

There were several limitations in this study that should be recognized and improvements should be made if further studies are to be carried out.

The experiments were not carried out using physiological concentration of BSA and therapeutic concentrations of drugs. The ratio of BSA concentration to drug concentrations was significantly lower than the normal ratio [89, 90]. Saturable concentration-dependent protein binding depicted in our studies may not take place under normal therapeutic dosing of two drugs. As a result, the outcome of competitive binding between CPZ and VPL may not take place to a large extent at normal concentrations. Therefore, the validity of our finding may be limited. Furthermore, the number of binding sites and the association constants for both CPZ and VPL were not in any agreement with the related research conducted previously. This may possibly be due to limited number of data points obtained for Scatchard plots, resulting in a less accurate determination of binding parameters of drugs. Hence, further extensive research, using normal physiological and therapeutic concentrations of BSA and two drugs, should be conducted such that the pattern of binding chemistry of the two drugs to BSA can be more reliably determined [15, 28].

Site-specific displacement studies should be conducted to refine the location of competitive binding between CPZ and VPL. Individual protein binding of both drugs to the two specific binding sites on albumin, namely class V and Vl, could be studied using the respective site-specific probes erythromycin and imipramine [4, 91-94]. Therefore, the binding chemistry of CPZ and VPL would be more specific such that the rationale behind the displacement of CPZ by VPL would be better understood.

In our studies, results were accompanied by notable standard deviations. The low level of free drug concentrations present in testing samples was a cause of such fluctuation. Therefore, more wells should be combined to ensure measurable free drug concentrations that have a corresponding absorbance above the value of 0.1. However, combining more wells may increase the level of errors in the absorbance reading because final absorbance readings take into account of all possible errors from each of the wells. Hence, special care should be taken when filling up the wells to minimize the risk of obtaining error-prone readings.

Another major limitation of our studies is that protein binding of CPZ and VPL to alpha-1 acid glycoprotein (AAG) was not studied. Because of the basicity of both drugs, CPZ and VPL are known to bind significantly to AAG [28]. Binding pattern of CPZ and VPL to AAG might be different from that to albumin, possibly altering the overall degree of protein binding of both drugs. Additional studies of protein binding using AAG should hence be performed.

On the other hand, in our prediction of changes in volume of distribution, we assume that changes in fraction unbound of drug only take place in plasma but not in tissue. However, this over-simplified approach may underestimate or overestimate the overall effect on volume of distribution of drugs, limiting the validity of our finding. In order to overcome such limitations, alternative equations could be adopted, for example Oie-Tozer equation which takes into account other factors that affect the tissue distribution of drugs such as lipophilicity, degree of ionization and protein content in extravascular spaces [12, 15, 95-99].

Most importantly, our one-dimensional study assumes that the process of protein binding was the only determinant of the efficacy of CPZ. However, it failed to take into account that drug-drug interactions of CPZ with VPL also take place at other levels. For example, VPL may increase the CPZ penetration across the blood-brain barrier through inhibiting P-gp. The possibly enhanced CPZ penetration may override the displacement effect of CPZ by VPL, leading to an increase in efficacy of CPZ. Therefore, studies with more consideration of different dimensions should be undertaken such that the results obtained may draw a better conclusive prediction of any changes in the efficacy of CPZ in vivo.

5. Conclusion

The order of administrating CPZ and VPL appeared to have a significant impact on protein binding of CPZ to BSA but not on VPL. Protein binding of VPL seemed to be unaffected by the presence of CPZ. Our findings showed that VPL displayed a higher binding affinity for the common binding sites on BSA shared by CPZ and VPL. This competitive binding resulted in the reduction of protein binding of CPZ when CPZ and VPL were co-administered. The rationale behind the difference in protein binding of CPZ in two displacement studies remained unknown due to insufficient data. Further research, with more consideration of other drug-drug interactions, should be conducted to provide a more detailed analysis of the binding chemistry.

Our findings suggest that VPL should be orally administered prior to CPZ to achieve better clinical outcome. However, the impact on the pharmacological effects of CPZ in our studies may be mar