Overview of Sotrastaurin Clinical Pharmacokinetics

John M. Kovarik, PhD and Alan Slade, PharmD

T-cell signaling and PKC-beta and PKC-delta in B-cell signal-

Abstract: Sotrastaurin (AEB071) is an investigational immuno- suppressant that blocks T-lymphocyte activation through protein kinase C inhibition. It is currently in Phase II of clinical development for the prevention of acute rejection after solid organ transplantation. In renal transplant clinical trials, sotrastaurin has been administered at doses of 200 to 300 mg twice daily. Using a validated liquid chroma- tography method with tandem mass spectrometry, steady-state predose blood concentrations averaged approximately 600 and 900 ng/mL at these dose levels, respectively. Sotrastaurin is primarily metabolized through CYP3A4. There is one active metabolite, N-desmethyl-so- trastaurin, that is present at low blood concentrations (less than 5% of the parent exposure). The elimination half-life of sotrastaurin averages 6 hours. Clinical drug interaction studies to date have demonstrated that sotrastaurin increases the area under the concen- tration–time curve of everolimus 1.2-fold and of tacrolimus twofold. Conversely, sotrastaurin area under the concentration–time curve is increased up to 1.8-fold by cyclosporine and 4.6-fold by ketocona- zole. Blood samples from renal transplant patients receiving sotrastaurin were stimulated ex vivo by protein kinase C-dependent pathways. Inhibition of cytokine production, expression of CD69, and thymidine uptake served as biomarkers that demonstrated the ability of sotrastaurin to inhibit T-cell activation and proliferation at the doses used in these studies. Phase II trials have paired sotrastaurin with tacrolimus, mycophenolic acid, or everolimus. The clinical and pharmacokinetic results of these and upcoming trials will determine the optimal immunosuppressive regimen to benefit from sotrastaur- in’s novel mechanism of action and whether therapeutic drug mon- itoring will be beneficial.
Key Words: Sotrastaurin, protein kinase C, pharmacokinetics (Ther Drug Monit 2010;32:540–543)

PROTEIN KINASE C INHIBITION AND
DRUG DEVELOPMENT
Protein kinase C (PKC) is considered an attractive therapeutic target for the treatment of T-lymphocyte-mediated diseases.1 PKC isoforms regulate signal transduction pathways essential for the initiation and homeostasis of immune responses. Specifically, PKC-alpha and PKC-theta function in
ing.2 PKC-theta is selectively translocated to the site of contact between the T-cell and the antigen-presenting cell immediately after cell–cell contact thereby playing a pivotal role in T-cell activation3 and in interleukin-2 production.4 PKC-alpha in T- cells is necessary for proliferation and interferon-gamma production.5 PKC-delta and PKC-beta play roles in induction of tolerance and in antigen receptor function, respectively.6
Sotrastaurin (AEB071) is a low-molecular-weight, synthetic compound that potently and reversibly inhibits PKC-theta, PKC-alpha, and PKC-beta with lesser activity on PKC-delta (IC50 range, 1.0–2.1 nM).7 Sotrastaurin was tested first as monotherapy in a proof-of-concept study in patients with moderate to severe plaque psoriasis. At doses of 200 and 300 mg twice daily for 2 weeks, the psoriasis area severity index score was significantly reduced from baseline compared with placebo.8 This study demonstrated the potential of sotrastaurin for the treatment of T-cell-driven diseases and served as a springboard to enter into Phase II trials for the prevention of acute rejection in de novo renal transplantation.
Figure 1 shows the signaling pathways for T-cell activation. Signal 1 acts through the T-cell receptor and signal 2 through the coreceptor CD28. Cyclosporine and tacrolimus inhibit signal 1 through calcineurin, whereas sotrastaurin inhibits both signals 1 and 2 through PKC. Sotrastaurin therefore represents a new mechanism of action compared with currently marketed immunosuppressants used in organ transplant medicine. We present an overview of what is currently known about sotrastaurin clinical pharmacokinetics, the potential for pharmacokinetic drug interactions, biomarker responses to measure drug activity in clinical studies, and initial explorations for exposure–efficacy relationships in de novo renal transplant Phase II studies. Because some of these studies have not yet been fully evaluated or are ongoing, indi- vidual data from a renal transplant patient are used throughout this review to illustrate the in vivo effects of sotrastaurin. The index patient was enrolled in a clinical trial (registry number NCT00403416) and was randomized to receive 200 mg sotrastaurin twice daily with reduced-exposure tacrolimus (with basiliximab induction and corticosteroids) from the time of transplant to Month 3 in study part 1. Thereafter, tacrolimus was withdrawn from the regimen and replaced with mycophenolic acid in study part 2. The pharmacokinetic and biomarker data from this patient are representative of the

Received for publication June 24, 2010; accepted June 24, 2010. From Novartis Pharmaceuticals, Basel, Switzerland.
This work was presented at the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Pre-Congress Meeting in Montreal, Canada, October 2009.
Correspondence: John M. Kovarik, PhD, Novartis Pharma AG, Building 210.
427, 4002 Basel, Switzerland (e-mail: [email protected]). Copyright ti 2010 by Lippincott Williams & Wilkins
data from the study population as a whole.

SOTRASTAURIN
CLINICAL PHARMACOKINETICS
Sotrastaurin is administered orally twice daily in a cap- sule or tablet formulation. A validated liquid chromatography

540 Ther Drug Monit ti Volume 32, Number 5, October 2010

FIGURE 1. Schematic illustration of T-lymphocyte activation pathways and inhibition by immunosuppressants. T-cells are activated through signaling at the T-cell receptor (TCR) and through the coreceptor CD28. Cyclosporine and tacrolimus inhibit calcineurin (CN) and sotrastaurin inhibits protein kinase C (PKC). Both lead to an inhibition of interleukin-2 (IL-2) production through nuclear activation factors. IL-2, in turn, is necessary for T-cell proliferation.

method with tandem mass spectrometric detection is used to measure sotrastaurin concentrations in blood, plasma, and urine. To date, all clinical pharmacokinetic data have been derived from whole blood drug levels. Total drug exposure (area under the concentration–time curve [AUC]) is dose proportional over the single-dose range from 10 to 750 mg and over the multiple-dose range from 25 to 300 mg twice daily. Doses used in clinical trials in renal transplantation are 200 and 300 mg twice daily. A high-fat meal had a minor impact on AUC decreasing it by an average 17%. In clinical trials, it is recommended that patients administer sotrastaurin consis- tently either with or without food to avoid food-related fluc- tuations in drug exposure over time. Steady-state blood levels of sotrastaurin are reached within 2 days after initiating treatment and there is minimal drug accumulation from the first dose to steady state (accumulation ratio, 1.16). Figure 2 shows the sotrastaurin concentration–time profiles over the 12-hour dose interval for the index renal transplant patient at the end of Week 1 posttransplant when receiving 200 mg sotrastaurin twice daily with tacrolimus and corticosteroids. The steady-state predose C0 was 198 ng/mL, Cmax was 1770 ng/mL, tmax was 2 hours postdose, and AUC ss was 7376 ngtih/mL. Subsequent concentration–time profiles ex- hibited a narrower peak-trough fluctuation with Cmax around 1100 ng/mL and C0 approximately 400 ng/mL. Nonetheless, overall drug exposure was stable over the full study course as reflected in the AUC of 8715 ngti h/mL at Month 3 (study part 1, sotrastaurin with tacrolimus) and 7626 and 9468 ngti h/mL at Months 4 and 6 (study part 2, sotrastaurin with mycophenolic acid), which were similar to the value in Week 1.
Renal excretion of sotrastaurin is negligible. Rather, sotrastaurin is primarily metabolized to inactive metabolites

FIGURE 2. Steady-state concentration–time profiles over a 12- hour dose interval for sotrastaurin in a representative renal transplant patient receiving 200 mg sotrastaurin twice daily with reduced-exposure tacrolimus at Week 1 and Month 3 and with mycophenolic acid at Months 4 and 6. Basiliximab was given as induction therapy and corticosteroids were used throughout the 6-month observation period.

with the exception of N-desmethyl-sotrastaurin, which has similar potency as sotrastaurin in the mixed lymphocyte reaction assay. Because blood levels of N-desmethyl-sotras- taurin are low compared with those of the parent drug (generally less than 5%), they likely do not contribute to efficacy to a clinically relevant extent. In the index renal transplant patient, steady-state N-desmethyl-sotrastaurin CO was 5 ng/mL, Cmax was 23 ng/mL, and AUC was 122 ngti h/mL. In this case, total N-desmethyl-sotrastaurin exposure was less than 2% compared with that of the parent compound. The half-lives of sotrastaurin and N-desmethyl-sotrastaurin could not be robustly determined in the index renal transplant patient as a result of the short period of blood sampling (12-hour dose interval), but average 6 and 12 hours based on data from single-dose studies in healthy subjects.

POTENTIAL FOR DRUG INTERACTIONS
In vitro experiments using human liver microsomes and CYP-specific inhibitors indicated that sotrastaurin is a substrate of CYP3A4 and 2C8 and of ABCB1 transporter (P- glycoprotein). Conversely, sotrastaurin can inhibit the metab- olism of CYP1A2, 2C8, and 3A4 substrates and the transport of ABCB1 substrates in vitro.
Although the clinical drug interaction study program for sotrastaurin is ongoing, several single-dose studies in healthy subjects have been completed and begin to sketch out sotrastaurin’s drug interaction profile. Sotrastaurin’s potential to be the victim of drug interactions is illustrated by the fol- lowing: 1) sotrastaurin AUC was increased weakly by 1.2-fold and 1.8-fold when the strong ABCB1 inhibitor cyclosporine was coadministered at a low and a high dose, respectively9; and 2) sotrastaurin AUC was increased moderately by 4.6-fold when the strong CYP3A4 inhibitor ketoconazole was coad- ministered.10 The potential of sotrastaurin to perpetrate drug interactions on CYP3A4/ABCB1 substrates is illustrated by the weak 1.2-fold increase in everolimus AUC11 and the

Kovarik and Slade Ther Drug Monit ti Volume 32, Number 5, October 2010

moderate twofold increase in tacrolimus AUC.12 Clinical drug interaction studies to assess the ability of sotrastaurin to alter the pharmacokinetics of CYP1A2 and CYP2C8 substrates and the ability of a CYP3A4 inducer to alter the pharmacokinetics of sotrastaurin are planned.

BIOMARKERS OF DRUG ACTIVITY IN
CLINICAL TRIALS
Biomarkers to investigate PKC-dependent T-lymphocyte functions were measured ex vivo in healthy subjects receiving sotrastaurin alone and in renal transplant patients receiving sotrastaurin in the context of multidrug immunosuppressive regimens. In these studies, venous blood samples were collected predose and at multiple time points postdose. As biomarkers of T-cell activation, the percent of T-lymphocytes producing interleukin-2 and tumor necrosis factor alpha (IL-2/TNF) and the percent of T-lymphocytes expressing the activation marker CD69 were quantified by flow cytometry after ex vivo stimulation of blood by the PKC activator phorbol-13- myristate-acetate and the costimulator anti-CD28 antibody. As a biomarker of lymphocyte proliferation, [3H]-thymidine incorporation into T-cells was measured by scintigraphy after ex vivo stimulation of blood with phytohemoagglutinin A.
In healthy subjects, all three biomarkers demonstrated dose- and concentration-dependent inhibition over the sotrastaurin single dose range of 100 to 750 mg. The nadir measurement (maximum inhibition) generally occurred 2 to 6 hours postdose roughly coinciding with the peak drug concentration in blood and recovery back to predose values was dose-dependent (approximately 8 hours after 100 mg and 24 hours after 750 mg). These three biomarkers were also measured in the index renal transplant patient before trans- plantation and then at predose and 2 and 6 hours postdose at Week 1 and Months 3, 4, and 6 (at the same visits as for pharmacokinetics). At the pretransplant baseline visit, 0.6% of T-cells were IL-2/TNF-positive, 51% of T-cells were CD69- positive, and thymidine uptake was 15 3 103 counts per minute. At Week 1 posttransplant, the nadir measurements postdose were 0% IL-2/TNF-positive T-cells, 18% CD69- positive T-cells, 0.5 3 103 counts per minute thymidine uptake; these nadirs represented 100%, 65%, and 97% reduction from pretransplant, respectively. Inhibition remained relatively stable over the course of the study. For example, at the last visit at Month 6, the nadir measurements postdose were 0.1% IL-2/TNF-positive T-cells, 7% CD69-positive T-cells, and 0.1 3 103 counts per minute thymidine uptake; these nadirs represented 83%, 86%, and 99% reduction from pretransplant, respectively.

CLINICAL EXPERIENCE IN RENAL TRANSPLANTATION
To date, three clinical trials in de novo renal transplantation have been initiated. In these studies, sotrastaurin has been combined with tacrolimus (trial registry number NCT00403416), mycophenolic acid (NCT00492869), or everolimus (NCT00820911). All trials also used basiliximab induction and corticosteroids. Sotrastaurin was administered at fixed doses of
either 200 mg or 300 mg twice daily. Pharmacokinetic blood sampling was an integral component in all studies whereby steady-state predose trough concentrations (C0, ss) were obtained in all patients at each protocol-scheduled visit and concentration–time profiles over a dosing interval were obtained in a subset of patients at selected visits. Immunosuppressant drug concentrations were determined at the end of the trial at a central laboratory by validated liquid chromatography methods with tandem mass spectrometric detection.
Combination with standard-exposure or reduced-exposure tacrolimus yielded excellent efficacy, whereas efficacy was suboptimal when combined with mycophenolic acid.13 In both cases, we explored for exposure–response relationships using the median-effect analysis14 to relate sotrastaurin predose trough blood levels to freedom from treated biopsy-proven acute rejection. Given the low incidence of treated biopsy-proven acute rejection when sotrastaurin and tacrolimus were combined,13 there was no clinically useful relationship between either sotrastaurin or tacrolimus trough blood levels versus efficacy.15 Although overall efficacy was inadequate when fixed- dose sotrastaurin was combined with mycophenolic acid,16 we noted retrospectively that freedom from treated biopsy-proven acute rejection increased with increasing sotrastaurin trough level: 73% at CO less than 602 ng/mL, 80% at CO from 602 to 1005 ng/mL, and 88% at CO greater than 1005 ng/mL. The study combining sotrastaurin with everolimus is currently in progress and will be explored for exposure–efficacy relation- ships after completion.

SUMMARY
The clinical development of sotrastaurin is currently in Phase II for the prevention of acute rejection after renal transplantation. The basic clinical pharmacokinetics of sotrastaurin have been determined in healthy subjects and renal transplant recipients. Clinical drug–drug interaction studies have shown so far that sotrastaurin exposure is increased weakly (less than twofold) to moderately (two- to fivefold) by strong ABCB1 or CYP3A4 inhibitors. Sotrastaur- in increases exposure of CYP3A4 substrates also in the weak to moderate range. Biomarker measurements have docu- mented that the sotrastaurin doses used in clinical trials elicit measurable inhibition of PKC-dependent T-cell activation and proliferation ex vivo. Further studies are needed to determine the optimal immunosuppressive regimen to benefit from sotrastaurin’s novel mechanism of action and whether therapeutic drug monitoring will be beneficial.

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