Tamoxifen citrate salt, internal standard verapamil hydrochloride, potassium dihydrogen phosphate, magnesium chloride and ortho-phosphoric acid (H₃PO₄) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Isoflurane was purchased from Cenvet (Kings Park, WA, Australia) while gavage needles (18G) were purchased from Livingstone, NSW, Australia. An 18G needle was used in these studies because it is thinner than 16G and can easily pass through the oesophagus without causing any damage to mucosal membrane and distress to the animal. The high-performance liquid chromatography (HPLC) grade acetonitrile, methanol and hexane were from Analytical Science (Sydney, NSW, Australia). Irradiated rat pellet diet was purchased from Speciality Feeds (Glen Forrest, WA, Australia).

The commercial PSP extract (ONCO-Z^®) was supplied by PuraPharm International (H. K.) Ltd., Hong Kong. The PSP extract is made from the fruiting body of dried 100% wild C. versicolor extracted in deionised water and contains absorbable peptidoglycan (APG). The standardised extract (340 mg) is equivalent to 2.83 g of dried C. versicolor and comes from a USP-accredited biological procedure for standardisation involving a freeze-drying process. The extract was verified by the U.S. Pharmacopoeia (The U.S. Pharmacopeia, 2019) in August 2009 and comprises of at least one peptide linked glucan, with glucose molecules as a monosugar connected by a 1→3 linkage. The crude extract (molecule weight of 0.5–40 kDa) comprises of an average 4.7% peptide/protein composition, 55% neutral sugar and 4.8% uronic acid. The purified extract has a molecular weight of 0.3–5 kDa (average 2.6 kDa) and is highly water soluble. The amino acid sequence of the protein/peptide moiety was determined to be Asp-Cys-Pro-Pro-Cys-Glu (Chow and Chu, 2006). To maintain verification status, the manufacture of the extract must continue under the same conditions. A voucher specimen was deposited at NICM Health Research Institute, Western Sydney University, Australia (Batch no: A1301475). A partial structure of the PSP polysaccharide component is proposed in a previous study (Hor et al., 2011).

The PSP extract was prepared in distilled water at a concentration of 170 mg/mL and administrated according to the animal weight (2 mL/kg). The concentration was chosen to reflect a high dose (340 mg/kg) and was under the considered lethal dose of more than 5,000 mg/kg (Hor et al., 2011).

Tamoxifen was prepared at a concentration of 15.2 mg/mL dissolved in water and administrated by oral gavage according to the animal’s weight (2 mL/kg). As tamoxifen is given orally to patients, oral gavage allowed the drug to be delivered directly to the stomach and this minimises the error associated with free feeding. The tamoxifen dose was chosen to keep the plasma concentrations above the detection limit (Choi and Kang, 2008).

Adult female Sprague-Dawley rats (over 12 weeks of age; 215–340 g) were bred in-house in the School of Medicine Animal Housing Facility, Western Sydney University. This rat species, gender and methods described has been used in other tamoxifen drug interaction studies, and this information provides a basis for this in vivo study (Shin et al., 2006; Choi and Kang, 2008; Kim et al., 2010; Kim et al., 2020). Throughout the experiment, three rats were weight-matched and randomly housed in the facility in Green Line individually ventilated cages (IVC) (Techniplast, United States), in a temperature-controlled room (22 ± 3°C), 50–60% relative humidity, under a 12 h light-dark cycle. The animals were acclimatised for at least 1 week with a normal diet and water ad libitum. During the study, rat observations included posture, coat hair, activity, movement, breathing, alertness, appetite changes/vomiting, dehydration, eyes, nose, faeces, and abnormal bleeding. This was monitored daily using an animal health monitoring sheet. Initial and final weights were recorded. The rats were anaesthesised using a high dose of isoflurane (and/or ketamine and xylazine as an option) to bring them into a surgical plane of anaesthesia and bled completely by cardiac puncture. Isoflurane anaesthesia has shown no significant effects on plasma metabolites (Davis, 1996).

The pharmacokinetic interaction study consisted of two main parts: a single oral dose of tamoxifen and repeated dosing of tamoxifen (to reflect chronic dosing and achieve steady state), with PSP extract fed orally for 7 days in both scenarios.

Thirty rats were randomly divided into six groups of five rats each. There were two control groups: Grp 1a control (untreated, only water) and Grp 1b PSP-treated control (PSP extract administered orally daily at 340 mg/kg for 7 days), with serum collected on day 8.

There were two tamoxifen control groups: Grp 2a single tamoxifen oral dose (30.4 mg/kg tamoxifen citrate equivalent to 20 mg/kg tamoxifen base) on day 8 with serum collected on the same day; and Grp 2b repeated tamoxifen oral dosing (20 mg/kg per day) for 13 days with serum collected on day 13. In humans, the steady state for tamoxifen is achieved in about 4–6 weeks (Kisanga et al., 2004). In rats, it has been shown that steady state is reached in 3 days (Lien et al., 1991). Thus, 13 days for Grp 2b depicted the chronic administration of tamoxifen in clinical practice, and this timeframe allowed for any biochemical changes to occur.

There were two PSP treatment with tamoxifen groups: Grp 3a single tamoxifen oral dose (20 mg/kg) on day 8, pre-treated with PSP extract administered orally at 340 mg/kg daily for 7 days (to emulate pre-treatment of the natural product) with blood sampling done on day 8 to discern PSP’s effect on a single tamoxifen dose; and Grp 3b repeated tamoxifen oral dosing (20 mg/kg per day) for 12 days plus PSP extract administered orally daily at 340 mg/kg from days 5 to 11, with blood sampling done on day 12. For Grp 3b, the repeated daily dosing of tamoxifen reflects chronic administration in clinical practice, with PSP taken daily during active treatment. Tamoxifen was administered in the morning, whilst PSP extract was administered in the afternoon to provide a time gap between the two interventions.

Three rats were sampled, in the morning, at any one time. This allowed the blood sampling time frames to be achieved. The rats were fasted for at least 12 h prior to blood sampling to ascertain whether the extract continued to have any effect on the drug. During blood sampling, each rat was anesthetised by inhalation with isoflurane which allowed quicker and cleaner access for blood sampling and rapid recovery. Blood samples (up to 0.3 mL) were collected from the lateral saphenous vein at 0, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, and 24 h. Multiple small samples are unlikely to cause hypovolaemia to the animal (Diehl et al., 2001). The blood was centrifuged after sampling, and the serum samples were stored at −80°C until analysis.

All the animal experiments were carried out in accordance with the strict guidelines of the Animal Research Regulation 2005 (NSW) and the Australian code of practice for the care and use of animals for scientific purposes. Approval was endorsed by the Animal Ethics Committee of Western Sydney University [Animal Research and Teaching Proposal (ARTP) Approval Number: A9873] using the principles of replacement, reduction and refinement. The reporting of the results followed the Animal Research Reporting of In Vivo Experiments (ARRIVE) guideline (Kilkenny et al., 2010).

Chromatography was performed with a Shimadzu LC-20AT delivery unit consisting of a DGU-20A degassing solvent delivery unit, SIL-20A auto injector, CTO-20A column oven and SPD-A detector (Kyoto, Japan). Chromatographic separation was achieved by a Synergy MAX-RP-80A (4 µ, 150 × 4.6 mm) (Phenomenex, Torrance, CA, United States) attached to a 1 mm Optic-guard C-18 pre-column (Optimize Technologies, Alpha Resources, Thornleigh, Australia) in ambient temperature. The isocratic mobile phase, using a modified method as described previously (Antunes et al., 2013; Yang et al., 2013), comprised of 50 mM potassium phosphate buffer (pH 2.15) and acetonitrile (55:45, v/v) using verapamil hydrochloride as the internal standard. The mobile phase was delivered at a flow rate of 1 mL/min. The eluent was monitored at 280 nm ultra-violet detection.

A stock solution of tamoxifen citrate (2 mM: equivalent to 0.371 mg/mL of tamoxifen) was prepared in methanol and was further diluted with methanol to give a series of working solutions of 0.12, 0.23, 0.46, 0.93, 1.86, 3.71, and 7.42 μg/mL. A stock solution of verapamil (50 μg/mL) was prepared in methanol. Both stock solutions were stored at −20°C and working solutions were freshly prepared from the prepared stock solution when required. Standard curves were prepared by adding known concentrations of tamoxifen and verapamil to drug-free rat plasma.

The LC method was validated according to the International Council for Harmonisation guideline (The International Council for Harmonisation 1996). Low, middle and high concentrations of tamoxifen samples (0.023, 0.186, and 0.742 μg/mL, respectively) were prepared by spiking 10 µL of the tamoxifen working solutions (0.23, 1.86, and 7.42 μg/mL) into 100 µL of blank rat serum and stored at −20°C. Serum calibration standards (0.012–0.742 μg/mL) were freshly prepared for each analysis by spiking 10 μL of working solutions of tamoxifen into 100 μL of blank pooled rat serum which was pre-thawed at room temperature.

Briefly, 100 µL of serum samples were spiked with 5 µL of verapamil (final concentration: 2.5 μg/mL) after the deproteinisation with 100 µL of acetonitrile. The samples were extracted twice with n-hexane (900 µL) and the organic layer was removed and dried at 30°C for 30 min under vacuum in a Speed Vac concentrator (Thermo Scientific, United States). The dried samples were reconstituted with the mobile phase (90 µL), and 30 µL was injected into HPLC system.

The pharmacokinetic parameters of tamoxifen were determined by a non-compartmental analysis using PKSolver (Zhang et al., 2010). Maximum serum concentrations (C_max) and time to reach maximum concentration (T_max) for both the single and repeated tamoxifen dosing experiments were determined by visual inspection of the serum concentration vs. time curve. The elimination constant rate (k_el) was estimated by semi-log linear regression of the terminal slope, and elimination half-life (t_1/2) was estimated by ln2/k_el. Area under the curve (AUC) and area under the first moment curves (AUMC) from 0 to last observed concentration (AUC_0-t and AUMC_0-t, respectively) were determined by the linear trapezoidal method.

For the 24 h time point of the rat’s serum, 23 biochemical serum parameters, incorporating hepatic [e.g., alkaline phosphatase (ALP), alanine aminotransferase (ALT), total protein, albumin, globulins, total bilirubin], renal (e.g., creatinine, urea, calcium, phosphate) and cardiac (e.g., cholesterol, triglycerides, bile acids) diagnostics, were examined for all six rat groups. The samples were analysed by an external veterinary diagnostic laboratory [Veterinary Science Diagnostic Service (VSDS), University of Queensland, Australia] who were blinded to the control/experimental groups.

The depletion of tamoxifen alone and in the presence of PSP extract was performed using female rat liver microsomes (Sigma-Aldrich, Australia) in vitro using methods as previously described (Yang et al., 2013). The rat model emulates the metabolism of tamoxifen in humans (Lien et al., 1991). Briefly, 5 µL of tamoxifen (0.5 mM dissolved in 25% acetonitrile) was preincubated with or without the PSP extract (10, 50, and 100 μg/mL) in 0.5 mL of 0.1 M phosphate buffer (pH 7.4) containing a nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system (1 mM NADP+, 0.8 U glucose-6-phosphate dehydrogenase and 3 mM glucose-6-phosphate; Promega, Sydney, NSW, Australia) and 3 mM magnesium chloride in an open air shaking water bath at 37°C for about 3 min. Acetonitrile in the final incubation was 0.25%. After the preincubation, the enzymatic reaction was then initiated by adding 0.5 mg/mL of female rat hepatic microsomes. During the incubation, 100 µL aliquots were removed at time (t) = 0, 20, and 30 min. Each extracted aliquot was mixed with 200 µL of ice-cold acetonitrile to deactivate the enzymatic reaction. The resultant mixture was vortexed and centrifuged at 14,000 × g for 10 min, and the supernatant (30 µL) was directly injected to the HPLC system for analysis as described in the previous section. The overall in vitro intrinsic clearance (Cl_int) was estimated by the substrate depletion method using in vitro t_1/2 approach (Obach, 1999) rather than the product formation method (Rane et al., 1977) as it is known that tamoxifen undergoes multiple CYP-mediated metabolism. Briefly, using the AUC of tamoxifen at t = 0 as 100% of substrate, the AUC of the other time points were converted to a percentage of the substrate remaining, plotted as natural log of remaining drug versus incubation time, and the slope of the regression line, represented as depletion rate of constant (-k), was used for estimation of the in vitro t_1/2 by the following equation: i n   v i t r o   t 1 / 2 = − 0.693 / k

Subsequently, in vitro Cl_int was calculated by following formula: ( 0 . 693 / in   vitro   t 1 / 2 ) × ( mL   incubation   volume / mg   microsomal   protein ) .

The data was expressed as the means ± standard error of mean (SEM) of the separate experiments using Windows Excel. The pharmacokinetic data was compared using unpaired t-test with two-tailed p value. The author (BK) who completed the pharmacokinetic data was blinded to the actual experimental groups. The biochemical parameters were compared by one-way analysis of variance (ANOVA), followed by Tukey’s/Dunnet’s method for multiple comparisons using the program GraphPad Prism (San Diego, United States). The author who performed the biochemical statistics was not blinded to the groups. p values less than 0.05, 0.01, and 0.001 were considered statistically significant. All pharmacokinetic parameters are expressed as mean ± standard deviation, except for the elimination t_1/2 (harmonic mean ± pseudo-standard deviation).

The retention times for both tamoxifen and verapamil were approximately at 7.9 and 2.2 min, respectively. The total run time for each sample was 16 min. The concentrations of pharmacokinetic samples were determined by non-weighted least square linear regression of the serum calibration curve prepared daily and ranged from 0.0116 to 0.742 μg/mL (r ² ≥ 0.998), where peak area ratios of tamoxifen to verapamil were used. The calibration curve was y = 4.042x-0.015 (based on the average of three calibration curves). Based on the calibration curves, a lower limit of quantification was estimated at 0.0232 μg/mL where the precision and accuracy were within accepted criteria [< 15% of coefficient of variation (CV) and within 20% of nominal concentration, respectively]. A test for lack of fit revealed no departure from linearity for the calibration curve (F = 0.186; df = 5, 14; p = 0.186), indicating that the linear model was valid. For quality control (QC) samples (0.0232, 0.0927, and 0.742 μg/mL), the intra- (n = 3) and inter-day (n = 3 days) precision, expressed as CV, ranged from 2.81 to 4.69% and 8.53–14.10%, respectively. Intra-day and inter-day accuracy of QC samples were within 95.08–115.43% and 84.00–117.55%, respectively.

The single tamoxifen oral dose serum concentration-time profile with and without PSP extract is shown in Figure 1. It is observed that the single tamoxifen oral dose plus PSP extract showed a different serum-concentration profile compared to tamoxifen alone. The curve for tamoxifen plus extract exhibits a slower rate to its peak concentration in the first 10 h compared to the steeper single tamoxifen oral dose curve. The error bars indicate greater variability in the time points for the single dose which may indicate the extract’s control on tamoxifen’s absorption.

The pharmacokinetic parameters for the single tamoxifen dose are shown in Table 1. The AUC, C_max and t_½ for tamoxifen were comparable to literature values in other tamoxifen interaction studies (Shin et al., 2006; Choi and Kang, 2008; Kim et al., 2010). However, there was a significant difference between T_max values between the groups, with PSP extract increasing the value by three times. Although there was no significant difference between the two regimens for maximum concentration (C_max) and area under the serum concentration-time curve from 0 to 24 h (AUC_0–24h) (p > 0.05), PSP extract pretreatment showed a tendency to reduce both the C_max and AUC_0–24h of tamoxifen. PSP extract pretreatment also reduced the rate and extent of absorption of tamoxifen as seen from the decreased slope of the absorption phase, thus affecting the C_max and AUC_0–24h. Limitations in sampling time points precluded further calculations of the parameters using the PKSolver software.

The repeated tamoxifen oral dosing serum concentration-time profile with and without PSP extract is shown in Figure 2. The curve for tamoxifen plus extract shows an increased peak after 10 h.

The tamoxifen repeated-dose pharmacokinetic parameters are shown in Table 2. As for the single dose experiments, the results showed a significant difference (p < 0.05) between the T_max values, with T_max almost doubling with the administration of PSP extract. There was no significant difference between the tamoxifen only and tamoxifen plus PSP extract for the other measured parameters. As a comparison, there was no significant difference between the T_max values of the single and multiple dose experiments for tamoxifen alone and for tamoxifen and PSP extract (p > 0.05).

The results of the biochemical rat serum parameters for control (i.e., untreated and PSP-treated), single and repeated tamoxifen oral dosing with or without PSP extract are shown in Table 3. Twenty-three parameters were examined. There were no significant differences for the PSP-treated, single tamoxifen oral dose and single tamoxifen oral dose pretreated with PSP extract compared to untreated control (p > 0.05) for all the parameters tested.

There was no significant difference between the untreated control and repeated tamoxifen oral dosing for sodium (Na), magnesium (Mg), phosphate (PO₄), total bilirubin (TBIL), triglycerides (TRIG), urea (UREA), creatinine (CREAT), glucose (GLUC), non-esterified fatty acids (NEFA), and globulin (GLOB). Although there was a tendency to increase bile acids (BA) with repeated tamoxifen, there was no significant differences compared to untreated control. Repeated tamoxifen oral dosing plus PSP extract tended to reduce BA level compared to untreated control. For cholesterol (CHOL), repeated tamoxifen oral dosing showed comparable levels to untreated control, however, repeated tamoxifen oral dosing plus PSP extract showed reduced cholesterol compared to untreated control (p < 0.05).

For potassium (K; p < 0.01), chlorine (Cl; p < 0.001), creatine kinase (CK; p < 0.05), alanine aminotransferase (ALT; p < 0.05) and aspartate aminotransferase (AST; p < 0.001), there was a significant increase between untreated control and repeated tamoxifen oral dosing levels. Repeated tamoxifen oral dosing plus PSP extract reduced these values compared to untreated control.

For carbon dioxide (CO₂), there was a significant decrease between untreated control and repeated tamoxifen oral dosing (p < 0.05). Repeated tamoxifen oral dosing plus PSP extract increased this to untreated control levels.

For calcium (Ca), there was a significant decrease between untreated control and repeated tamoxifen oral dosing levels (p < 0.05). Although there was no significant difference, repeated dose tamoxifen plus extract increased this value slightly.

For TP and ALB, there was a significant decrease between untreated control and repeated tamoxifen oral dosing levels (p < 0.05 and p < 0.001, respectively). However, there was no significant difference between repeated tamoxifen oral dosing plus PSP extract and repeated tamoxifen oral dosing.

Alkaline phosphatase (ALP) results showed that repeated tamoxifen oral dosing was not significantly different to untreated control (p < 0.05). However, there was a significant increase between untreated control and repeated tamoxifen oral dosing plus PSP extract (p < 0.001). Although there was an increase in gamma-glutamyl transpeptidase (GGT) with tamoxifen (and a concomitant decrease with tamoxifen plus PSP extract), GGT values were not significantly different between the groups.

In this study, all microsomal incubation conditions were within the linear range of the reaction rate, and greater than 10% of the substrate was depleted compared to the initial substrate amount (t = 0 min) (Yang et al., 2013). Using substrate depletion constant rates (Obach and Reed-Hagen, 2002), the estimated K_m value [substrate concentration at half the maximum velocity (V_max)] of tamoxifen was 18 μM, indicating that the tamoxifen concentration (5 µM) used in this study was acceptable.

Tamoxifen was stable with microsomal incubation that lacked the NADPH regenerating system indicating that the depletion of tamoxifen in female rat hepatic microsomes was NADPH dependent. Compared with the in vitro Cl_int of the control (12 ± 4.2 μL/min/mg microsomal proteins), there were no significant differences observed for the in vitro Cl_int of tamoxifen when co-incubated with different PSP extract concentrations (10, 50, and 100 μg/mL) (Table 4). Consequently, it is unlikely that the PSP extract (between 10 and 100 μg/mL) will significantly inhibit the metabolism of tamoxifen which is pharmacologically mediated via CYP activity.

The disappearance of tamoxifen was also examined in male rat microsomes, and it was found that the clearance rate of tamoxifen was about three-fold faster than the female rat microsome. As in the female microsomes, PSP extract did not appear to alter the depletion of tamoxifen in the male rat microsome (data not shown).