Pharmacokinetics of high-dose simvastatin in refractory and relapsed chronic lymphocytic leukemia patients
To evaluate the pharmacokinetics of simvastatin at the maximum tolerated dose (MTD) of 7.5 mg/kg, twice daily, in the context of a pilot trial enrolling patients with recurrent and refractory chronic lymphocytic leukemia.
Patients received simvastatin orally at MTD for 7 days during a 21-day cycle for 6 cycles. Blood samples were collected during cycle 1. Simvastatin lactone and carboxylate concentrations were measured in plasma and peripheral blood mononuclear cells (PBMCs) using a validated HPLC–MS/MS assay.
Patients accrued to this study showed high variability in their exposure to simvastatin. Exposure was dose proportional (AUC and Cmax) as compared to those receiving standard hyperlipidemia therapy. Peak plasma concentrations ranged from 0.08 to 2.2 and from 0.03 to 0.6 μM for simvastatin lactone and carboxylate, respectively.
Our study shows that when simvastatin is administered at its MTD, only low micro-molar concentrations are achieved in plasma and PBMCs, which is consistent with the results observed in previous studies with lovastatin, but far lower than the concentrations required for anticancer effects in vitro. However, whether simvastatin at its MTD can confer therapeutic benefits to patients still remains to be determined.
KeywordsSimvastatin Pharmacokinetics High-dose Leukemia
Over the past two decades, statins have been used safely and effectively for the treatment for hypercholesterolemia. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme at the top of the mevalonate pathway, which is responsible for cholesterol synthesis . In addition to cholesterol, the mevalonate pathway yields other downstream products such as isoprenoids, dolichol, and ubiquinone , which are critical components for a wide range of cellular metabolic and signaling processes. In particular, isoprenoids (farnesyl pyrophosphate and geranylgeranyl pyrophosphate) are crucial for the anchoring of small GTPases, such as RAS and RHO family proteins, to the cell membrane . Membrane attachment allows the subsequent activation of these proteins, which mediate intracellular signaling for several downstream survival and proliferation processes .
In this context, statins have been tested for their potential use as anticancer agents in several tumor types. Several reports have shown that statin-mediated inhibition of isoprenoid synthesis disrupts small GTPases localization to the membrane and is likely the underlying mechanism for the in vitro observed antitumor activity [3, 4]. Notably, these reports have demonstrated that statins display anticancer activity only at concentrations higher than those observed in plasma of patients being administered typical doses associated with hyperlipidemia therapy [3, 4, 5].
Therefore, clinical investigators were prompted to study the safety and tolerability of high-dose statins in cancer patients. In a lovastatin phase-I study in patients with solid tumors, the maximum tolerated dose (MTD) of lovastatin was 25 mg/kg daily . In that study, the peak plasma concentrations of lovastatin were in the range of 0.1–3.9 μM, which are comparable to its IC50 values in glioma cells (0.2–2 μM). However, in a subsequent phase-I/II study of high-dose lovastatin in patients with malignant glioma, only one partial response and one minor response were observed out of nine patients . In the case of simvastatin, a phase-I study was conducted in patients with myeloma or lymphoma, and the maximum tolerated dose (MTD) of oral simvastatin was determined to be 7.5 mg/kg twice daily, for 7 days . However, the study design did not include pharmacokinetics, and it remains unknown whether simvastatin at high doses can reach the concentrations required for the antitumor activity observed in vitro. In a subsequent phase-II study, simvastatin at MTD was given for 7 days followed by rapid intravenous infusion of vincristine, adriamycin, and dexamethasone orally (VAD) on days 7–10. High-dose simvastatin failed to reverse clinical resistance to VAD chemotherapy in myeloma patients . Authors of this study attributed the limited efficacy of simvastatin to the short period of treatment as well as the treatment strategy. However, failure to reach therapeutically effective concentrations might be a possible explanation of these unsuccessful clinical results. Here, we report the pharmacokinetics of simvastatin given at MTD in patient with recurrent and refractory chronic lymphocytic leukemia (CLL).
Simvastatin for in vitro studies was purchased from Toronto Research Chemicals Inc. (North York, Canada). Ammonium acetate was from VWR (West Chester, PA, USA). HPLC grade acetonitrile and diethyl ether were obtained from Sigma-Aldrich (St Louis, MO, USA). Lovastatin (Alexis Biochemicals, San Diego, CA, USA) and glacial acetic acid were from Fisher Scientific (Fair Lawn, NJ, USA). Heparinized BD Vacutainer Cell Preparation Tubes were purchased from Becton to Dickinson (Franklin Lakes, NJ, USA).
Pharmacokinetic study design
Serial blood samples (8 mL) were collected in heparinized BD Vacutainer Cell Preparation Tubes during cycle 1 at predose, 15 min and 1, 2, 3, 6, 8, 12, and 24 h and at predose on day 7. Upon collection, samples were immediately centrifuged (1,800×g for 30 min at room temperature) to separate plasma and peripheral blood mononuclear cells (PBMCs) from whole blood. Top layer (plasma and PBMCs) was collected and centrifuged at 1,500 rpm for 5 min to separate plasma from PBMCs, and samples were stored at −80 °C until analysis. An LC–MS/MS method was developed and validated to measure simvastatin and its carboxylate form in human plasma and PBMCs . Detailed information about the study design and method of analysis can also be found in supplement materials and methods.
Pharmacokinetic data analysis
Plasma concentrations versus time data were evaluated by compartmental modeling using Phoenix WinNonlin 6.2 (Pharsight Corporation, Mountain View, CA, USA). Various compartment models were tested to determine the most appropriate model. The plasma pharmacokinetic parameters of simvastatin lactone and carboxylate, including the maximum observed plasma concentration (Cmax) and time to Cmax (Tmax), terminal phase elimination half-life (t1/2) and the area under the plasma concentration–time curve (AUC) from time 0 to time of the last measurable concentration (AUCt), were also calculated by non-compartmental analysis. PBMCs concentration of simvastatin was calculated based on the cellular volume of the collected PBMCs sample with considering the volume of CLL cell = 200 fL. CLL cell count in each sample was determined through measuring the protein concentration of the sample relative to those obtained from standard CLL samples with known cell count.
Plasma and PBMCs pharmacokinetics
Three patients were accrued between July 2009 and January 2011. The first participant accrued, remained on treatment for three cycles of therapy before experiencing disease progression. Of note, this participant reported an initial decrease in constitutional symptoms including fatigue, and the clinical investigators noted a substantial decrease in the patient’s palpable adenopathy. Due to the waxing and waning nature of CLL, it is unknown whether the change in symptoms and adenopathy is attributable to the effect of simvastatin. The subsequent two participants experienced progression of leukemia during their first cycle of therapy and were subsequently removed from therapy. One participant experienced grade 1 limb pain as the only toxicity attributed to the treatment.
Pharmacokinetic parameters in plasma for simvastatin lactone and carboxylate after oral administration of MTD of simvastatin to CLL patients (n = 3)
AUC12 (μM h)
As shown in Fig. 1, patient 2 had higher plasma concentrations of both forms of simvastatin relative to the other two patients. Simulation of multiple dosing of simvastatin based upon the final PK model for 6 days revealed no marked accumulation of either simvastatin lactone or carboxylate after the second dose or at day 6 in the three patients (Fig. 1). The model predicted clearance also showed that there was a fivefold variation in the estimated lactone clearance (i.e., CL/F) (Table 1).
Similarly, simvastatin lactone and carboxylate were measured in PBMCs, and as shown in supplement Fig. 1, patient 2 had the highest concentrations, as compared to the other patients, which correlated with their plasma concentrations. Notably, it was only the simvastatin lactone that was detectable in the PBMCs of these three patients.
Beyond their cholesterol lowering effect, several reports have shown that statins have anticancer properties in different tumor types. This effect is believed to be mediated through the inhibition of isoprenoid synthesis and the subsequent deactivation of small GTPases, which are involved in regulating multiple cellular functions including proliferation and survival. However, these in vitro studies have shown that statins display their anticancer activity at micro-molar concentrations that cannot be achieved with typical anticholesterolemia doses. This provided the rationale for testing the safety and tolerability of statins at high doses in cancer patients. Simvastatin was well tolerated, and its MTD was 7.5 mg/kg twice daily for 7 consecutive days in a 21-day cycle. This pilot clinical study demonstrated that simvastatin administered at its MTD achieved low micro-molar concentrations (Cmax), which, based on in vitro and ex vivo evidence, are unlikely to be effective.
Initial attempts to fit the pharmacokinetic data to a two-compartment model, as previously reported, were not successful in two of three patients. A four-compartment model was found to better characterize the data obtained from these patients. However, in order for the model to fit the data, several assumptions, based on previous pharmacokinetic publications [11, 12], had to be made. Furthermore, although the model was adequately fit to data from day 1, it did not predict the modest accumulation of either form of simvastatin, which was observed on day 7. This observed accumulation may be due to slight saturation of metabolic and/or transport processes following the repetitive administration of high-dose simvastatin.
Our results are in accord with previous studies of high-dose lovastatin. In that study, patients with solid tumors were administered lovastatin, and the MTD was 25 mg/kg . As a part of the study, pharmacokinetics were conducted, and peak plasma lovastatin concentrations ranged from 0.1 to 3.9 μM with an average concentration 2.32 μM. These in vivo concentrations were found to be comparable to those effective in glioma cells in vitro. Nonetheless, this approach did not show success in the clinic where high-dose lovastatin exhibited limited efficacy in glioma patients in a subsequent phase-II trial . Similarly, simvastatin at its MTD (7.5 mg/kg, given orally, twice daily) failed to reverse clinical resistance to VAD chemotherapy in myeloma patients . The short period of treatment (7 days) as well as the treatment strategy was denoted as potential factors that contributed to the unsuccessful clinical results. However, a longer period of treatment (21 days) with lovastatin at 7.5 mg/kg/day did not show any objective responses in patients with head and neck squamous cell carcinoma or cervical cancer . Recently, a phase-II study found no evidence of beneficial effect of high-dose simvastatin on disease markers in multiple myeloma patients . The investigators of those two clinical studies of high-dose simvastatin assumed that simvastatin reaches similar concentrations in plasma to those achieved with lovastatin. This was a reasonable assumption, since the pharmacokinetics of these two statins are similar at lower doses . Although few patients were accrued in our study, results from the plasma analysis of simvastatin after high dose support this assumption. The simvastatin plasma concentrations in our patients showed similar, but relatively lower Cmax range (0.08–2.2 μM) compared to lovastatin (Cmax: 0.1–3.9 μM). This higher Cmax range of lovastatin is likely within the interpatient variability range and may also be attributed to the difference in dosage regimen. Lovastatin dosing was more frequent (6.25 mg/kg four times daily) relative to simvastatin (7.5 mg/kg twice daily). Overall, our study was in agreement with previous lovastatin studies that reported low micro-molar concentrations in plasma after administering high doses [6, 16]. Moreover, the high interpatient variability seen in these studies was also observed among patients enrolled in our study, which may be attributed to several factors including differences in metabolism, as well as differences in oral absorption, due to efflux or incomplete dissolution of the high doses administered.
The few aforementioned phase-II trials of high-dose statins were initiated considering the fact that plasma peak concentrations achieved by these doses have been shown to be effective in vitro. However, the limited activity of statin seen in these clinical trials addresses some concerns about whether statins at high doses are really achieving therapeutically effective concentrations at the relevant tissues. Several in vitro studies have reported that statins were effective against glioma and myeloma cells at low micro-molar concentration ranges 1–10 μM [17, 18] and 0.8–13.3 μM , respectively. Noteworthy is the fact that the primary cells collected from glioma and myeloma patients were found to be more resistant to statins compared to established cell lines. For example, lovastatin was found to inhibit the proliferation of primary cells obtained from patients with glioblastoma at 10–100 μM . Similarly, lovastatin inhibited cell proliferation of primary myeloma cells at IC50 values ranging from 6 to 63 μM . Together, these observations indicate that the maximum plasma concentrations achieved with high-dose statins are only approaching the lower range of effective concentrations required for anticancer activity in primary myeloma and glioma cells. Therefore, comparing effective in vitro concentrations of statins in established cancer cells with those seen in patients may not be a valid approach in these cases. In agreement with this observation, our in vitro data indicate that simvastatin induces apoptosis in primary CLL cells only at suprapharmacologic concentrations (~100 μM, data not shown), which are not attainable in vivo. This may in part explain the disease progression in the CLL patients treated with high-dose simvastatin in this study.
From another perspective, simvastatin carboxylate is known to be the active form that mediates the antitumor activity of simvastatin through the inhibition of the HMG-CoA reductase enzyme. In our study, simvastatin carboxylate was found to be present in plasma at lower concentrations compared to simvastatin lactone. Moreover, it was not observed [or below the detection limit 5 ng/mL (0.01 μM)] in CLL cells isolated from these patients, even at high level of exposure as in the second patient. The hydrophilic nature of the carboxylate form may have hindered its accessibility into CLL cells. In general, limited accessibility of the simvastatin active form to the tumor site may be considered a critical factor added to other factors that contribute to the poor response seen in all the previous clinical trials.
In conclusion, pharmacokinetic data in CLL patients showed that simvastatin administered at its MTD achieves plasma concentrations that are far lower than those shown to be effective ex vivo in primary CLL cells. In view of these data, the poor pharmacokinetic profile of simvastatin makes it unlikely to be successful as a sole therapy for treatment for CLL and perhaps other cancer types. Statins that have high bioavailability and long elimination half-life may be further considered for better clinical benefits in cancer therapy. Additionally, seeking rational synergistic combinations that include statins at concentrations achieved in vivo with other anticancer agents may provide an alternative approach for improving statin clinical activity.
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