Drug-drug interactions (DDI) occur when one drug (perpetrator drug) varies the plasma concentration and the biological outcomes of a drug (victim drug) (Hasnain et al., 2017). There are two types of DDIs, i.e., Pharmacokinetic drug-drug interactions (PK-DDIs) and Pharmacodynamic drug-drug interactions (PD-DDIs). PK-DDIs result from changes in plasma concentrations of a ‘victim’ drug caused by a ‘perpetrator’ drug altering the metabolism or transporter-mediated disposition of the victim drug. In particular, the cytochrome P450 (CYP) system, which is responsible for the metabolism of many drugs, can be influenced by other drugs leading to PK-DDIs. Induction of CYP enzymes can increase the metabolism and clearance of a victim drug, resulting in reduced plasma concentrations and potentially reduced efficacy. On the other hand, inhibition of CYP enzymes can decrease the metabolism and clearance of a victim drug, leading to increased plasma concentrations and potentially increased risk of adverse effects (Storelli et al., 2018). Drug transporters, such as P-glycoprotein (P-gp), multidrug resistance protein 2 (MRP2), and breast cancer resistance protein (BCRP), also play a significant role in drug absorption and excretion. Inhibition or induction of these transporters can affect the bioavailability and elimination of drugs, leading to PK-DDIs (Marchetti et al., 2007; Niwa and Hata, 2016). Other factors, such as age, gender, nutritional status, diseases, genetic polymorphisms, and ontogeny of metabolic enzymes, can also impact drug metabolism and contribute to PK-DDIs. For example, some drugs may have different pharmacokinetic profiles in elderly patients than in younger individuals due to age-related changes in drug metabolism. Understanding and predicting PK-DDIs are crucial in clinical practice to optimize medication therapy and prevent adverse effects. Healthcare professionals should be vigilant in considering potential interactions when prescribing or adjusting drug regimens, and patients should inform their healthcare providers about all the medications they are taking, including prescription, over-the-counter, and herbal products, to minimize the risk of PK-DDIs. Pharmacokinetic drug-drug interactions can be managed through appropriate drug selection, dosing adjustments, and close monitoring of drug concentrations and clinical response. In some cases, alternative medications with lower interaction potential may be chosen, or the timing of drug administration may be adjusted to minimize the risk of PK-DDIs. Overall, pharmacokinetic drug-drug interactions can significantly impact the safety and efficacy of medications by altering their absorption, distribution, metabolism, or excretion. Therefore, understanding the mechanisms and factors contributing to PK-DDIs is critical for healthcare professionals to make informed decisions in medication management, optimize patient outcomes, and minimize the risk of harm (Marchetti et al., 2007; Niwa and Hata, 2016; Hasnain et al., 2017; Storelli et al., 2018). Similarly, our study drug (Voriconazole) is a narrow therapeutic index drug; requiring close monitoring when administered with other drugs (Ashbee et al., 2013). Therefore, it is essential to characterize the PK-DDIs potential of Voriconazole with co-administered drugs.

Voriconazole synthetically derived from fluconazole antifungal agent (Wong‐Beringer and Kriengkauykiat, 2003), having a chemical composition [(2R, 3S) -2- (2, 4-difluorophenyl) -3-(5-fluora-4pyrimidinyl) -1- (1H −1, 2, 4-trizole-1-yl) -2-butanol] and has a broad spectrum (Greer, 2003; Herbrecht, 2004). Voriconazole is rapidly absorbed and has 96% oral bioavailability (B.A) (Geist et al., 2013; Hohmann et al., 2016). Voriconazole is highly metabolized by the hepatic enzyme CYP2C19 and forms a voriconazole-N-oxide as a major inactive metabolite; other metabolites formed are hydroxyl voriconazole and dihydroxy-voriconazole (Greer, 2003). Voriconazole shows the first-pass effect by primary systemic metabolism occurring by cytochrome-P450 enzymes, for example, CYP2C19, CYP2C9, CYP3A4, and CYP3A5. Up to 25% of metabolism occurs by Flavin containing mono-oxygenase FMO-1 and FMO-3 in enterocytes and hepatocytes (Yanni et al., 2008; Vanhove et al., 2017). Voriconazole is a potent inhibitor of CYP2C19, CYP2C9, CYP2B6, and CYP3A4 of hepatocytes and enterocyte enzymes (Jeong et al., 2009). Moreover, voriconazole is administered (oral or IV); its total dose has been excreted as metabolites (98%) within 48 h (Roffey et al., 2003). Renal and Biliary excretion of voriconazole (the metabolized form) is about 75%–80% and 20%–25%, respectively, while the remaining 2% is excreted in the urine in an unchanged form (15). Deliberating voriconazole pharmacokinetics and considerable inter-individual variability in drug disposition have been reported because, in drug disposition, genetic polymorphism of the metabolizing enzymes may have a starring role (Levêque et al., 2006; Hohmann et al., 2016). Voriconazole is also a substrate of p-glycoprotein (ABCB1) located at different sites (intestines and excretory organs) (Mikus et al., 2011). Allegra et al. reported that breast cancer resistance protein (BCRP1), multidrug resistance-associated protein (MRP2, also known as ABCC2), ABCG2, and solute carrier organic anion transporter (SLCO1B3, also known as OATP1B3) transporters might have a role in variation in voriconazole plasma-concentration in pediatrics (Allegra et al., 2018). Voriconazole is an inhibitor of several transporters like BCRP, p-glycoprotein, MRP (its other members MRP-1, MRP-2, MRP-4, and MRP-5), and bile salt export pump (BSEP) (Lempers et al., 2016).

Clarithromycin (6-O-Methylerthromycin) is a semi-synthetic macrolide antibacterial agent with a 14-membered ring (Alkhalidi et al., 2008). Clarithromycin is a frequently prescribed antibiotic drug nowadays. Clarithromycin is a substrate of several transporters (ABCB1, ABCC2, OATP2B1, and OATP1A2) located at different sites (intestinal, hepatic, and renal) (Peters et al., 2011). Clarithromycin is also an inhibitor of p-glycoprotein located at enterocytes (luminal), hepatocytes (canalicular), and renal (luminal) sites, as well as an inhibitor of OATP1B1 and OATP1B3 located at hepatocytes (sinusoidal) and intestine (Wakasugi et al., 1998; Niemi, 2007; Müller and Fromm, 2011). Clarithromycin is extensively metabolized by hepatic CYP3A4. Clarithromycin is an intense inhibitor of CYP3A4 and has a moderate inhibitory activity of CYP2C19, CYP2D6, and CYP1A2 enzymes present at the hepatic and intestinal level (Michalets, 1998; Furuta et al., 1999). Clarithromycin is a recognized inhibitor of CYP3A4, while many drugs are a substrate of this enzyme, so clarithromycin alters the AUC and plasma concentration of astemizole (Rodvold, 1999), cisapride (Haarst et al., 1998) and pimozide (Desta et al., 1999). As a result of PK-DDI, clarithromycin raises the AUC of these drugs (Michalets, 1998; Rodvold, 1999).

Clarithromycin is weakly basic in nature (Grübel and Cave, 1998), with 8.76 PKa (Nakagawa et al., 1992). Voriconazole exhibits a set of pKa values, i.e., basic-1.76 PKa value (Adams and Bergold, 2005; Adams et al., 2008) and acidic PKa values: 4.36 and 12.7 (Owens et al., 2000; Damle et al., 2011; Vanstraelen et al., 2015), respectively. In this viewpoint, the chemical nature as evident by pKa of both interacting drugs (voriconazole and clarithromycin co-administered simultaneously) make them candidates for possible potential PK-DDIs. Likewise, clarithromycin and voriconazole have 42%–72% (Langtry and Brogden, 1997) and 58% (Geist et al., 2013) protein binding, respectively. Clarithromycin is CYP3A4 (Gorski et al., 1998) and CYP2C19 (Furuta et al., 1999) enzyme inhibitor, and voriconazole is also a substrate (Vanhove et al., 2017) and inhibitor (Jeong et al., 2009) of these two enzymes; hence both candidate drugs share the same enzyme pathway. Being a substrate of the same enzyme and transporter, there is a likelihood of PK-DDI between voriconazole and clarithromycin. Enzyme CYP2C19 has genetic polymorphism making the population fall as poor, moderate, and extensive metabolizers (Bahar et al., 2017). Asian peoples are mostly poor CYP2C19 metabolizers, so that DDI may be possible in this region, and voriconazole may show variable C_max because of non-linearity (Mikus et al., 2011). Previously reported patterns of voriconazole-DDIs (Donnelly and De Pauw, 2004; Pasqualotto et al., 2010; Dolton et al., 2014; Bahar et al., 2017) and clarithromycin-DDIs (Michalets, 1998; Rodvold, 1999), as well as the PK parameter of both drugs, predicted that DDI might be possible. There is a possibility of co-administration of both drugs in certain clinical situations (Purkins et al., 2003a; Soler-Palacín et al., 2012; Mishima et al., 2017; Hirai et al., 2022). Therefore, we aimed to evaluate the interaction between voriconazole and clarithromycin in healthy Pakistani male volunteers. Till date, no study has been reported on assessing the effect of clarithromycin on the pharmacokinetic parameters of voriconazole.

In the current PK-DDI study of voriconazole with clarithromycin, we have enrolled more than 20-year aged healthy Pakistani male volunteers (n = 15). However, three out of these 15 volunteers later withdrew due to personal problems. Therefore, the DDI study was carried out on the remaining 12 volunteers (as presented in Figure 1), and these 12 subjects were selected according to mentioned criteria. Furthermore, the range of volunteers’ age, along with their mean with standard deviation (±SD), was 21–25 years and 23.3 ± 1.23 years, respectively, while the range of volunteer’s weight, height, and BMI, as well as their mean with ±SD, was 63–71 kg, 1.62–1.79 m, and 22.50–24.90 kg/m² and 67.51 ± 2.47 kg, 1.69 ± 0.04 m and 23.77 ± 0.91 kg/m², respectively. In addition, an isocratic, reversed-phase high-performance liquid chromatography ultraviolet/visible detector (RP HPLC UV-Vis) method was developed to analyze the voriconazole standard (stock-solution) and voriconazole in plasma samples. The method offered a simple liquid–liquid extraction LLE technique, which exhibited best recovery of voriconazole along with fluconazole, i.e., internal standard. Different experimental conditions were tried and ultimately, the best outcomes were accomplished utilizing C-18 Perkin-Elmer^® column with particulars of 150 mm length, 4.6 mm inner diameter and 5 μm particle size, utilizing mobile-phase of acetonitrile-water (ACN: H₂O) in a proportion of 60: 40 v/v, having a flow rate of 1.5 mL/min, and wavelength of 254 nm. All the analytes were observed to be separated in ≤7 min. The peak of voriconazole and fluconazole (internal standard) were visible at 5.25 and 4.20 min retention time, respectively. The correlation coefficient of voriconazole was observed to be 0.999, and average recovery (in percent) was 97.4%, whereas the relative standard deviation value was ≤2%. The lower limit of detection LLOD was 0.01 μg/mL, whereas lower limit of quantification LLOQ was 0.03 μg/mL, respectively. The results expressed that the adapted method of voriconazole has high recovery (Mushtaq et al., 2022). Further, semi-log and linear graphs of plasma concentrations of voriconazole were plotted as a function of time after administration of voriconazole alone and voriconazole along with clarithromycin, as graphically represented in Figure 2A and 2B. PK-Summit^® (version 2.0.2; PK Solutions) SPSS software (version 21.0; IBM Crop; SPSS^®; 2012), and Microsoft Excel were used to calculate mean with standard deviations, % difference, geometric mean ratio and confidence interval for all PK parameters of voriconazole, as summarized in Table 3.

Antibiotics and antifungals are sometimes administered in conjunction in clinical therapeutic settings (Purkins et al., 2003a; Soler-Palacín et al., 2012; Mishima et al., 2017; Hirai et al., 2022). For instance, voriconazole and clarithromycin are prescribed simultaneously to treat Invasive Pulmonary Aspergillosis. It is a serious and often life-threatening fungal infection that commonly affects immune-compromised patients, such as those with hematologic malignancies or undergoing solid organ transplantation (Soler-Palacín et al., 2012). Voriconazole is considered a first-line treatment for Invasive Pulmonary Aspergillosis, and clarithromycin may be prescribed concomitantly to treat bacterial coinfections or to provide additional coverage against atypical bacteria (Purkins et al., 2003c; Soler-Palacín et al., 2012; Xing et al., 2017). Another indication that voriconazole and clarithromycin may be prescribed together is in treating nontuberculous mycobacterial infections, particularly those caused by Mycobacterium Avium Complex (MAC). Clarithromycin is often used as part of the multidrug regimen for MAC infections, and voriconazole may be added in cases where there is coexisting fungal infection or suspected fungal coinfection (Purkins et al., 2003c; Xing et al., 2017). Likewise, voriconazole and clarithromycin are prescribed simultaneously in treating infectious endophthalmitis (Purkins et al., 2003c; Mishima et al., 2017; Xing et al., 2017). Further, both participating drugs share the same enzyme pathway, providing a basis for evaluating the PK-DDI behavior of voriconazole and clarithromycin. Non-linear pharmacokinetic behavior of voriconazole is providing a base for many DDIs (Brüggemann et al., 2009). Voriconazole is a CYP2C19 and CYP3A4 enzyme inhibitor and a substrate of these enzymes (Mikus et al., 2011). Clarithromycin is a substrate and potent inhibitor of CYP3A4. It also moderately inhibits the CYP2C19 (Furuta et al., 1999), i.e., a main metabolic enzyme of voriconazole.

A significant increase in the C_max value (52%), and also AUC^0-∞ value (21%) of voriconazole was observed after concomitant administration of clarithromycin, which is practically considered to be of clinical importance. The reason for this increment in AUC and C_max of voriconazole may be the inhibition of CYP3A4 and CYP2C19 enzymes by clarithromycin because clarithromycin is substrate and inhibitor of these enzymes (Furuta et al., 1999). Similar results were reported in many studies (Purkins et al., 2003b; Wood et al., 2003; Heinz et al., 2007; Andrews et al., 2008; Yasu et al., 2016) that demonstrated the effect of CYP2C19 and CYP3A4 inhibition has been evaluated over the PK of voriconazole and reported the increment of the AUC and C_max of voriconazole. Table 4 represents the increment in AUC and C_max of voriconazole due to inhibition of CYP isoenzyme by ethinyloestradiol and norethindrone (Andrews et al., 2008), cimetidine and ranitidine (Purkins et al., 2003b), omeprazole (Wood et al., 2003), pantoprazole (Heinz et al., 2007), lansoprazole (Yasu et al., 2016), esomeprazole (Bouatou et al., 2014), tacrolimus (Mochizuki et al., 2015), haloperidol (Motta et al., 2016), etravirine (Kakuda et al., 2013), azithromycin and erythromycin (Purkins et al., 2003d).

A decrease in apparent clearance and an increase in AUC were observed in our study. This interaction may be possible by two mechanisms; decreased metabolism and interaction at the transporter level. In-vitro data suggested that clarithromycin inhibitory concentration of CYP34A is 48% and CYP2C19, as well as 2C9 values are 11% and 4%, respectively (Obach et al., 2006). The pattern of clarithromycin predicted a slighter decrease in the metabolism of the CYP2C19 substrate (Obach et al., 2005). Voriconazole has a greater affinity for CYP2C19, so it is expected that less increment in C_max of voriconazole should be the outcome because CYP34A is not a primary elimination pathway (Obach et al., 2005; Obach et al., 2006). In comparison, clarithromycin has a 60%–70% potential to inhibit the CYP3A4 at the intestinal level (Obach et al., 2006; Galetin et al., 2007). Therefore, clarithromycin potentially inhibited the metabolism of pimozide and midazolam (substrates for the CYP3A4 activity), as reported by several researchers (Gorski et al., 1998; Desta et al., 1999).

In addition, clarithromycin is a potent CYP3A4 inhibitor (inhibition constant K i = 57.5 µM; K I = 13.2 µM; K i n a c t = 0.058/min) (Elsby et al., 2019). The Michaelis-Menten constant ( K m ) of voriconazole oxidase activity was 235 μM/L for CYP3A4 expressed in human CYP enzyme, which shows low affinity towards CYP3A4 (Mikus et al., 2006). Therefore, when the K m and K i values of these substances ( K m for CYP3A metabolism of voriconazole, 235 μM/L; K i for CYP3A4 inhibition by clarithromycin; K i = 57.5 µM) were taken into account, an interaction was expected with clarithromycin (Quinney et al., 2009; Burt et al., 2010).

Our results showed a 52% increase in plasma concentration might be because of decrease in the metabolism of voriconazole by clarithromycin. Nevertheless, the exact extent of DDI is not predictable because no in-vivo data show significant interaction (i.e., a significant increase in voriconazole plasma concentration) with another macrolide. The decrease in apparent clearance observed in our study may be because both interacting drugs are substrates and inhibitors of p-glycoprotein/ABCB1 transporter at intestinal, hepatic, and renal levels (Mikus et al., 2011; Müller and Fromm, 2011; Lempers et al., 2016). Clarithromycin has the potential to inhibit the various transporters because in-vitro data suggested that I C 50 values of clarithromycin for P-glycoprotein and MRP2 were 8.9 ± 0.5 µM and >50 μM, respectively (Vermeer et al., 2016). Interestingly, clarithromycin also has inhibitory potential against OATP transporter, e.g., I C 50   o f OATP1B1 and 1B3 are 5.3 ± 1.3 µM and 14 ± 2 μM, respectively (Müller and Fromm, 2011; Vermeer et al., 2016).

The equation t 1 / 2 = 0.693 E r a t e shows that half-life is inversely proportional to the elimination rate constant. Our study results show an increase in elimination rate constant (E_rate) from 0.09/hour to 0.11/hour, so half-life became reduced. It may be due to transporter involvement (Mikus et al., 2011; Müller and Fromm, 2011; Peters et al., 2011; Lempers et al., 2016; Allegra et al., 2018). The reduction in half-life could be due to changes in the elimination rate constant. Merely looking into the overlay graph of voriconazole alone and voriconazole concentration after administration of voriconazole along clarithromycin depicted that initially faster elimination rate and decreased in half-life, later on, elimination became slow, so overall decreased in the apparent clearance of voriconazole has been observed.

According to the equation, i.e., t 1 / 2 = 0.693   x   V d C l , a decrease in apparent clearance should generally increase the half-life. However, our results showed a decrease in the half-life of voriconazole. Similar results have been presented by Rengelshausen et al. (Rengelshausen et al., 2005), who have evaluated the impact of concomitantly administrated voriconazole with St. John’swort. They have observed a 20% reduction in the half-life of voriconazole. They suggested an increase in oral BA, a reduction in the distribution of voriconazole, and a short-term decrease of the systemic voriconazole distribution may be due to alteration in the transport process, and these are the probable mechanism of the reduction in the half-life of voriconazole besides decreased apparent clearance (Rengelshausen et al., 2005).

Our results showed a decrease in the apparent volume of distribution. According to the apparent clearance equation, i.e., Cl = KVd, when Vd decreases that leads to a decreased clearance value, provided that the elimination rate constant remains the same. A reduction in apparent clearance has been observed in our results. Wakasugi et al. (Wakasugi et al., 1998) have reported an increase in the AUC and C_max of digoxin on the concomitant administration of clarithromycin with digoxin by inhibiting p-glycoprotein (Wakasugi et al., 1998). Clarithromycin may reduce the voriconazole apparent clearance by competition and inhibiting the P-glycoprotein transporter. At the hepatocyte level, voriconazole and clarithromycin interaction may be possible because clarithromycin is an inhibitor of the SLCO1B3 (OATP1B3) transporter (Müller and Fromm, 2011), and voriconazole is a substrate of this transporter. An increase in AUC and C_max and reduced voriconazole apparent clearance may be due to the inhibition of hepatocellular uptake transporters (SLCO1B3/OATP1B3). Consequently, a reduced hepatic influx of voriconazole may lead to a reduction in metabolism (Allegra et al., 2018). A similar mechanism of DDI was presented between clarithromycin and paclitaxel involving hepatic OATP1B3 transporter inhibition. Efflux transporter, i.e., ABCC2, also known as MRP2, is common transporter for both interacting drugs (Peters et al., 2011; Allegra et al., 2018). Therefore, PK-DDIs may be possible among voriconazole and clarithromycin for competition for that common transporter. Interestingly, both interacting drugs (clarithromycin and voriconazole) are inhibitors of this transporter (Peters et al., 2011; Allegra et al., 2018).

A sharp difference in t_max from 2 h to 1.5 h (which means a 25% decrease) has been observed in our study. The decline in t_max may be due to the physiochemical nature of both interacting drugs. According to Biopharmaceutics Classification System (BCS), voriconazole (Kumar et al., 2014) and clarithromycin (Kristin et al., 2017) are both class-II drugs. Clarithromycin is a weak base with 8.87 pKa (Nakagawa et al., 1992; Grübel and Cave, 1998). Voriconazole exhibits basic and acidic pKa profiles, i.e., basic pKa: 1.76 (Adams and Bergold, 2005; Adams et al., 2008) and acidic pKa: 12.7 and 4.36 (Owens et al., 2000; Damle et al., 2011; Vanstraelen et al., 2015). Voriconazole nature may be a cause of this interaction. Clarithromycin is basic in nature and may provide a medium for the solubility of an acidic moiety of voriconazole. It is possible that acidic pKa is predominant at this stage, which is why t_max decreased and enhanced the dissolution. Likewise, clarithromycin is also a potent inhibitor of efflux transporter, i.e., p-glycoprotein (Müller and Fromm, 2011) and CYP450 enzyme system (CYP3A4 and CYP2C19) at the intestinal level (Furuta et al., 1999). So, these two reasons enhanced the absorption rate and decreased the t_max of voriconazole. Similar results have been reported by Rengelshausen et al., which demonstrated that St. John’swort decreased the t_max of voriconazole due to enhancing the dissolution rate (Rengelshausen et al., 2005). However, the present study was a single-dose study and single dose might not enhance the dissolution; further investigations are required to evaluate the PK profile of voriconazole in case of multiple dosing and a larger population.

A clinically significant PK-DDI of voriconazole and clarithromycin has been observed. In addition, we observed a 52% increase in the C_max of voriconazole during the co-administration of clarithromycin with voriconazole. Therefore, the dose of voriconazole must be adjusted to avoid severe and dangerous side effects like hepatotoxicity and neurotoxicity because voriconazole is a narrow therapeutic index drug.