Pharmacology, Pharmacokinetics, and Practical Applications of Bortezomib

Normal cellular function and homeostasis depend on precisely controlled intracellular processes, including the systematic and highly regulated degradation of proteins that control cellular division, growth, function, and death.[1-3] For example, regulation of the cell cycle depends on the orderly degradation of cyclins and inhibitors of the cyclin-dependent kinases.[1] If the systematic degradation of proteins is interrupted, regulatory proteins accumulate within the cell, creating an imbalance in the number of proteins required to elicit certain cellular functions, such as progression from the G1 to the S phase of the cell cycle or activation of signal transduction pathways.[ 4,5] In eukaryotic cells, the ubiquitinproteasome pathway (UPP), comprising a ubiquitin-conjugating system and the proteasome, is primarily responsible for the degradation of cellular proteins and plays an important role in many basic cellular processes.[ 5-7] The list of cellular proteins controlled by the UPP is growing rapidly and includes, in addition to the cell-cycle regulatory proteins (eg, cyclins, cyclin-dependent kinase inhibitors), proteins involved in chromatid separation, oncogenes and tumor suppressor genes, and transcriptional activators and inhibitors.[5,7-10] Another important function of the UPP is the selective removal of mutated and denatured or misfolded proteins, as well as proteins damaged by stress, oxidation, chemicals, or viral infection.[ 1,8,10] Aberrations in the UPP have been implicated in the pathogenesis of many diseases, including certain malignancies. For example, degradation of the p53 tumor suppressor gene or p27 inhibitor of cyclin-dependent kinases can promote tumorigenesis of various malignancies, including uterine, colon, breast, and prostate cancers.[ 1,7,8] Inhibiting the activity of the proteasome, one of the key constituents of the UPP, can block cellular growth and division, ultimately leading to cell death. Because the proteasome is responsible for degrading more than 80% of cellular proteins in eukaryotic cells, its inhibition would seem incompatible with life.[5] However, the results of preclinical and early-phase clinical studies show that proteasome inhibition (PI) can arrest the growth of tumor cells, induce apoptosis, inhibit angiogenesis, and increase the sensitivity of tumor cells to chemotherapy or radiation therapy, without having a major deleterious effect on nontumor cells.[3,11-13] Based on these results, a number of synthetic PIs have been developed and evaluated as antitumor agents. Bortezomib(Drug information on bortezomib) (PS-341, Velcade), the first proteasome inhibitor to be evaluated in humans, received US Food and Drug Administration (FDA) approval for the treatment of patients with multiple myeloma previously treated with two or more therapies and with disease progression on the last therapy.[5,14-17] This article summarizes the pharmacology, pharmacokinetics, and current practical applications of bortezomib. Proteasome Inhibition To understand more clearly the mechanism of action of bortezomib (Figure 1), a basic understanding of the proteasome's role in the UPP and the consequences of PI are required.[ 1,18] Ubiquitin-Proteasome Pathway
Protein degradation by the UPP involves two discrete and successive steps: (1) tagging of the substrate (protein to be destroyed) by covalent attachment of multiple ubiquitin molecules, and (2) degradation of the tagged protein by the 26S proteasome.[ 7,8] Ubiquitin molecules are attached to the target protein by the sequential activity of three enzymes: E1, E2, and E3. E1, the ubiquitinactivating enzyme, activates the ubiquitin molecule through an adenosine(Drug information on adenosine) 5'-triphosphate (ATP)-dependent reaction and transfers it to one of many different E2 or ubiquitin-conjugating enzymes. The ubiquitin molecule is then transferred to the target protein in a step that requires the E3 enzyme (ubiquitin protein ligase). Eukaryotic cells contain hundreds of E3 enzymes, each with the ability to recognize different degradation signals on proteins. Ubiquinated proteins are then recognized and degraded by the 26S proteasome.[ 1,6-9] The 26S proteasome is a multifunctional proteolytic complex that consists of a proteolytic core particle, the 20S proteasome, and two 19S regulatory particles.[7] The 20S core particle consists of four stacked rings: two identical outer rings (α rings) and two identical inner rings (β rings). The α and β rings are composed of seven distinct subunits, giving the 20S complex the general structure of α _1-7 β_1-7 β _1-7 α _1-7. The proteolytic sites are localized on the β1, β2, and β5 subunits of the two inner β rings. Each end of the 20S core is capped with a 19S regulatory particle, which recognizes polyubiquinated proteins, cleaves the polyubiquitin chain from the target protein (the polyubiquitin chain can then be disassembled by deubiquitinating enzymes and recycled into the UPP), unfolds proteins that would be unable to fit through the narrow proteasomal channel, and opens the channel in the α ring to permit entry of the target protein into the proteolytic chamber. The opening of the channel requires metabolic energy, and each 19S regulatory particle contains six different ATPase subunits that provide energy upon hydrolysis by ATP. After the protein is degraded by the 20S core particle, short peptides are released into the cytosol of the cell. Because the active sites (β subunits) of the proteasome are confined to the inner cavity of the 20S core particle, uncontrolled degradation of cellular proteins cannot occur.[1,2,6-9] The beta subunits of the 20S core particle contain three types of proteolytic sites: chymotrypsin(Drug information on chymotrypsin)-like sites, trypsin-like sites, and caspase-like sites.[1,6,9] These sites differ in their specificity for the types of protein residues (eg, hydrophobic residues and acidic residues) at which they cleave. Although the 26S proteasome has several active sites, inhibition of all three sites is not required to significantly reduce protein processing. Specific inhibition of the chymotrypsin-like site by bortezomib significantly reduces protein processing (Ki = 0.6 nM). Most inhibitors of chymotrypsin-like sites are more highly hydrophobic and cell-permeable than inhibitors of the trypsin- or caspase-like sites, which contain charged residues; therefore, most proteasome inhibitors act predominantly on the chymotrypsinlike sites and to a much lesser degree on the other two sites.[1] Proteasome Inhibition
Various natural and synthetic proteasome inhibitors-all of which bind to and directly inhibit the active sites within the 20S core particle of the proteasome-have been identified.[ 1,5,11-13] Impeding the degradation of regulatory proteins through PI results in accumulation of several important regulatory proteins, including the inhibitor of nuclear factorkappa beta (NF-κB), IκB, the p53 tumor suppressor gene, the p21 and p27 cyclin-dependent kinase inhibitors, and the bax protein.[1,5,13,19- 22] Accumulation of these proteins leads to decreased NF-κB activity, increased p53-mediated transcription of genes important in apoptosis and dysregulation of the cell cycle, increased p21- and p27-mediated induction of cell cycle arrest, and promotion of apoptosis by inhibition of Bcl-2 by bax.[1,5,13,19-22] Proteasome inhibition also downregulates the p44/42 mitogen-activated protein kinase (MAPK)-induced signals required for tumorigenesis.[1,5] As a result of these and other incompletely understood effects of PI, proteasome inhibitors inhibit tumor cell proliferation, induce apoptosis, and inhibit angiogenesis. As mentioned previously, the results of several preclinical studies have shown cancer cells to be more sensitive to the effects of PI than are normal cells. For example, patientderived chronic lymphocytic leukemia cells are about 10 times more sensitive to PI than are normal human lymphocytes.[20] Although the biologic basis for the enhanced susceptibility of cancer cells to PI has not been fully elucidated, several hypotheses exist, including a greater sensitivity of rapidly proliferating cells (eg, tumor cells) to PI and more efficient uptake and slower inactivation of proteasome inhibitors by tumor cells.[1,5] Other theories focus on the deregulation of various UPP functions during the malignant transformation of a cell. For example, low levels of the bax proapoptotic protein resulting from an upregulation of UPP activity have been associated with higher Gleason scores in prostate cancer patients.[22] Proteasome inhibition blocks the degradation of this protein, resulting in higher intracellular levels and increased apoptosis.[22] In addition to producing antitumor effects, proteasome inhibitors sensitize both chemosensitive and chemoresistant cancer cells to conventional chemotherapy.[ 5,12,13,18,19,23,24] For example, the combination of bortezomib with irinotecan(Drug information on irinotecan) (Camptosar) was more effective in inhibiting tumor growth in mice than either irinotecan or bortezomib alone.[23] Combined with subtoxic doses of bortezomib, melphalan(Drug information on melphalan) (Alkeran), doxorubicin(Drug information on doxorubicin), and mitoxantrone(Drug information on mitoxantrone) (Novantrone) exhibit cytotoxic effects on chemoresistant multiple myeloma cell lines at drug concentrations 100,000- to 1,000,000-fold lower than concentrations required for cytotoxicity in the absence of bortezomib.[24] Proteasome inhibitors also play a role as radiation therapy sensitizers. In a mouse colon cancer model, a single dose of radiation therapy and bortezomib produced significantly lower tumor volumes and increased apoptosis rates compared with radiation therapy alone.[25] These combination therapies did not increase cytotoxic or radiotoxic effects on normal bone marrow cells in healthy, cancer- free individuals. The mechanisms by which proteasome inhibitors reverse chemotherapy or radiation therapy resistance are not completely understood, although downregulation of NF-κB has been shown to play an important role in abrogating drug resistance.[5,23] For example, NF-κB activity in multiple myeloma cell lines resistant to melphalan, mitoxantrone, and doxorubicin is greater than NF-κB activity in nonresistant multiple myeloma cell lines; treatment with subtoxic doses of bortezomib attenuated NF-κB activity, sensitizing the resistant cells to treatment with these agents.[22] Proteasome inhibitors may also downregulate other resistance pathways, including the p44/42 MAPK pathway, which is activated by certain chemotherapy agents, such as the taxanes and anthracyclines. In a murine xenograft model of breast cancer, proteasome inhibitors have been shown to block doxorubicin-mediated activation of the p44/42 MAPK pathway, which correlates with increased apoptosis and antitumor efficacy.[5] Proteasome inhibitors have shown a broad spectrum of activity in preclinical models. The dipeptide boronic acid bortezomib is a specific and reversible inhibitor of the chymotrypsin- like activity of the 26S proteasome. While many other proteasome inhibitors have been synthesized and tested in preclinical models, bortezomib is the only one to be clinically evaluated in cancer patients and approved for clinical use.[1,5,13-17] Pharmacology of Bortezomib Bortezomib is a potent, selective, and reversible inhibitor of the chymotrypsin- like activity of the 26S proteasome (see Figure 1). Preclinical and early-phase study results revealed that bortezomib was active against a broad range of hematologic and solid tumors, with tolerable effects on normal tissues.[3-5,11-14] The results of a phase I dose-determining study by Aghajanian and colleagues[3] showed that PI increases with increasing doses of bortezomib, with approximately 65% PI occurring after administration of the manufacturer-recommended dose of 1.3 mg/m². Maximum inhibition of 20S activity occurs within 1 hour after bortezomib administration; 20S activity returns toward baseline within 72 to 96 hours.[26,27] No significant difference in the mean percentage of PI was observed with administration of subsequent doses on days 4, 8, and 11, suggesting that 72 hours between administration is sufficient for recovery of proteasome function in normal tissues.[3] Pharmacokinetics The pharmacokinetics of bortezomib have not been fully characterized in multiple myeloma patients.[16] The pharmacokinetics have been investigated in two phase I studies in patients with solid tumors receiving combination therapy of bortezomib and irinotecan or gemcitabine(Drug information on gemcitabine) (Gemzar).[28,29] After intravenous (IV) bolus administration, bortezomib quickly distributes into tissues from the plasma.[17,27,28,30] The distribution half-life is less than 10 minutes, followed by a long elimination half-life (> 40 hours).[27] In animal studies using radiolabeled bortezomib, bortezomib was rapidly distributed into nearly all tissues, with the exception of adipose tissue and certain tissues in the brain protected by the blood-brain barrier.[30] Following extensive tissue distribution of radiolabeled bortezomib, a slow terminal elimination rate was observed, with only 65% (females) to 85% (males) of the total dose recovered from monkeys after 144 hours.[30] Plasma protein binding of bortezomib is considered moderate (approximately 83%) and was not shown to be concentration dependent over the concentration range studied (100 to 1,000 ng/mL).[17] The results of in vitro studies suggest that bortezomib is metabolized primarily through oxidative deboronation (the removal of boronic acid from the parent compound), which can be mediated by multiple cytochrome P450 system isoenzymes, including 3A4, 2C19, 1A2, 2D6, and 2C9.[16,17,31] Deboronation produces two inactive enantiomers that subsequently undergo further metabolic processing and are eliminated by both renal and hepatic routes.[30,32] More than 30 inactive metabolites have been identified in animal and human studies. Pharmacokinetics studies of bortezomib in patients with renal or hepatic insufficiency have not been completed; however, studies evaluating bortezomib in these patient populations are under way with the National Cancer Institute's Organ Dysfunction Group.[17,33] Clinical trials included patients with creatinine clearance values ranging from 13.8 to 220 mL/min.[17] No correlation between creatinine clearance and maximum PI at 1 hour, the incidence of grade 3 or 4 adverse effects, or discontinuation rates have been observed.[ 33] Patients with reduced renal function have displayed response and treatment discontinuation rates comparable to those in patients with more normal renal function and were able to receive a comparable number of bortezomib doses.[33] The pharmacokinetics in patients either undergoing hemodialysis or with a creatinine clearance value less than 13 mL/min have not been completely described.[17] The appropriate dosage of bortezomib in patients who were more than 30% above their ideal body weight was calculated based on average body weight ([actual body weight - ideal body weight]/2); however, the effectiveness of this method for determining an appropriate dose in obese patients is unknown.[34] Bortezomib use has not been evaluated in pediatric patients, although a pharmacokinetics study of bortezomib administered to pediatric patients is under way.[17] Practical Applications Based on the results of two phase II clinical trials, the SUMMIT and CREST trials, bortezomib received accelerated FDA approval on May 13, 2003, for the treatment of multiple myeloma patients whose disease has progressed after they have received at least two prior conventional therapies.[ 16,17,35] Studies evaluating bortezomib as first- and second-line therapy for multiple myeloma patients, including a phase III, multicenter, randomized trial comparing bortezomib with high-dose dexamethasone(Drug information on dexamethasone) in relapsed multiple myeloma patients, are under way.[14,16,35] Additional phase I or II studies evaluating bortezomib alone or combined with standard chemotherapy agents as multiple myeloma treatment and as treatment of solid tumor and other hematologic malignancies have shown promising results.[36-42] For a more indepth discussion of the clinical uses of bortezomib, refer to the article entitled "Discovery, Development, and Clinical Applications of Bortezomib" found in this supplement. Because bortezomib is a novel, first-in-class proteasome inhibitor approved for use in relapsed or refractory multiple myeloma, clinicians prescribing or monitoring bortezomib therapy should be educated about its effects in humans. Although additional studies are needed to define more clearly the effects of bortezomib administered either alone or in combination with other antitumor agents for various cancers, the results of phase I and II studies have provided useful information about monitoring the toxicity of bortezomib in relapsed or refractory multiple myeloma patients whose disease has relapsed or whose disease is refractory to conventional therapies. Adverse Effects
In the SUMMIT and CREST phase II trials, the most common adverse effects in multiple myeloma patients receiving bortezomib 1.3 mg/m², included fatigue (65%), nausea (64%), diarrhea (51%), thrombocytopenia (43%), anorexia (43%), peripheral neuropathy (37%), vomiting (36%), pyrexia (36%), anemia (32%), peripheral edema (25%), and dyspnea (22%).[16,17] Table 1 lists the severe (grades 3 and 4) adverse effects observed in these trials.[16,17] Most toxicities were mild to moderate in severity (grades 1 or 2) and did not require discontinuation or delay of bortezomib therapy. Treatment was withheld in patients experiencing grade 3 or higher nonhematologic toxicities or grade 4 hematologic toxicities.[14] Retreatment with a 25% dose reduction (ie, reduced from 1.3 to 1.0 mg/m² or from 1.0 to 0.7 mg/m²; doses below 0.7 mg/m² were not permitted) was allowed in patients who experienced a lessening in the severity of the adverse effect to a grade 1 or lower level.[14] Bortezomib-related toxicities that required treatment discontinuation, including peripheral neuropathy (5%), thrombocytopenia (4%), fatigue (2%), and diarrhea (2%), developed in 18% of patients.[17] In another phase II trial in mantle cell lymphoma patients receiving bortezomib, Assouline and colleagues[ 43] reported five cases of severe fluid retention in patients with baseline dyspnea or peripheral edema. Two patients died: one died of grade 4 acute vascular leak syndrome and the other died of progressive disease with severe edema. The other three patients experienced dyspnea and peripheral edema or hypoxia and peripheral edema. Based on these results, the authors amended their study to exclude patients with baseline dyspnea or fluid retention. Interestingly, less than 5% of multiple myeloma patients enrolled in the SUMMIT and CREST trials experienced grade 3 or 4 dyspnea or edema; presumably these patients showed no signs of fluid retention at study entry.[17] Nevertheless, patients with preexisting fluid retention, especially in the presence of dyspnea or hypoxia, should not receive bortezomib therapy, and patients should be instructed to report any signs of fluid retention promptly to their caretaker. Because of the potential need for dosage adjustment or requirement for premedication, several adverse events associated with bortezomib merit further discussion, including peripheral neuropathy, hypotension, thrombocytopenia, and gastrointestinal effects.

• Peripheral Neuropathy-Based on pooled data from the SUMMIT and CREST trials, treatment-emergent peripheral neuropathy-primarily sensory neuropathy characterized by burning or painful dysesthesias, paresthesias, or numbness-was observed in 35% of patients receiving bortezomib therapy.[44] Of the patients who experienced bortezomibinduced peripheral neuropathy, more than 70% had previously received neurotoxic therapies; additionally, more than 80% of patients reported symptoms of peripheral neuropathy at baseline.[17,44] Patients with these baseline symptoms were at greater risk of developing grade 3 or 4 peripheral neuropathy during bortezomib therapy.[ 44] Only two patients without baseline peripheral neuropathy symptoms developed grade 3 peripheral neuropathy, suggesting that the incidence of peripheral neuropathy may be lower in ongoing and future studies evaluating patients with earlierstage disease and who have received no or minimal previous neurotoxic therapies.[44] In patients experiencing grade 3 or 4 peripheral neuropathy requiring treatment discontinuation, symptoms disappeared or lessened in 37% of patients during treatment; however, the remaining patients experienced symptoms throughout and after discontinuing bortezomib therapy. Partial or complete reversal of symptoms occurred in 71% of patients; in 40%, symptoms resolved during bortezomib therapy and in 32% after therapy discontinuation. For patients who experienced resolution or improvement in peripheral neuropathy after completing therapy, the median time to resolution was 47 days (range: 1-529 days) after the last dose of bortezomib. Treatment-emergent peripheral neuropathy required therapy discontinuation or dosage reductions in only 5% and 12% of patients, respectively.[44] In an open-label extension study, patients from the SUMMIT and CREST trials who benefitted from bortezomib therapy were allowed to continue receiving bortezomib and were assessed for an average of 24.4 additional weeks (patients were assessed for 8 cycles, or 24 weeks, during the SUMMIT and CREST trials).[45] Evidence of cumulative or permanent toxicity, including peripheral neuropathy, after prolonged bortezomib exposure (median, 45 weeks; maximum, 99.9 weeks) did not exist. Early diagnosis of bortezomib-induced peripheral neuropathy and bortezomib dosage adjustments may prevent development of severe neuropathies; therefore, patients should be closely monitored for and instructed to report new or worsening symptoms of neuropathy (eg, increasing pain, numbness).[17] Additionally, the dosage should be adjusted in most patients experiencing painful peripheral neuropathy during bortezomib therapy (Table 2).[17] Data regarding the outcome of peripheral neuropathy in patients receiving bortezomib are limited, but analyses of follow-up data will be used to determine the pathophysiology and reversibility of peripheral neuropathy in these patients.[17,44]
• Hypotension-Orthostatic/postural hypotension occurred in 12% of multiple myeloma patients enrolled in the SUMMIT and CREST trials.[17] Grade 3 hypotension occurred in 4% of patients; grade 4 hypotension was not observed. None of these patients had evidence of orthostatic hypotension at baseline; however, approximately 50% had preexisting hypertension and 33% had symptoms of peripheral neuropathy. Four percent of patients experienced hypotension and a concurrent syncopal episode.[17]
Risk factors for the development of hypotension after bortezomib therapy include (1) history of syncope, (2) concomitant use of medications known to lower blood pressure (ie, antihypertensive agents), and (3) dehydration.[ 17] Hydration status should be assessed and corrected, if necessary, before and throughout bortezomib therapy, especially in patients experiencing nausea and/or vomiting. Additionally, patients receiving antihypertensive medications should be closely monitored to determine if antihypertensive medication dosage adjustment is necessary. Mineralocorticoids were effective in minimizing the hypotensive effects of bortezomib therapy in some patients.[17] Finally, patients and/or caregivers should be instructed to report signs or symptoms of hypotension (eg, lightheadedness, dizziness, syncope) immediately to a healthcare professional, maintain adequate hydration, and exercise caution when operating machinery, including automobiles. In patients experiencing grade 3 hypotension, the bortezomib dose should be discontinued until symptoms resolve, at which time a 25%- reduced dose of bortezomib may be implemented (see Table 2).[17]
• Thrombocytopenia-Patients receiving bortezomib 1.3 mg/m² in these trials experienced a median 60% decrease in their baseline platelet count during therapy regardless of initial baseline platelet count, baseline serum myeloma paraprotein (M-protein) level, or degree of multiple myeloma bone marrow involvement.[ 46] The onset of thrombocytopenia most commonly occurred after cycles 1 or 2 and continued throughout therapy.[17] Platelet counts typically reached a nadir on day 11 and rose to a normal count by day 21. Cerebral and gastrointestinal hemorrhages secondary to bortezomib- induced thrombocytopenia were rarely reported.[17] Patients with a baseline platelet count of less than 70,000/μL had an increased risk of developing grade 4 thrombocytopenia.[ 46] Furthermore, patients with greater bone marrow involvement (ie, > 50% plasma cells) or higher Mprotein levels (> 31 g/L) usually had lower baseline platelet counts and lower platelet count nadirs. Thrombocytopenia caused by bortezomib therapy presumably results from an inhibition of thrombopoiesis, an NF-κB-dependent process, rather than direct bone marrow toxicity; therefore, supportive care, rather than discontinuation of bortezomib therapy, may be adequate for controlling bortezomib's effects on platelet production.[46] Platelet counts should be monitored throughout bortezomib therapy, and therapy should be discontinued in patients with platelet counts less than 25,000/μL (grade 4 thrombocytopenia) until the platelet count returns to normal.[17] Bortezomib can be reinitiated at a 25%-reduced dose when platelet counts return to baseline levels. Patients and/or caregivers should be educated about the risks of bleeding and instructed how to manage a bleeding episode.
• Gastrointestinal Effects-Gastrointestinal adverse effects, including nausea, vomiting, diarrhea, constipation, and/or anorexia, are common in patients receiving bortezomib therapy. Nausea, vomiting, and diarrhea should be anticipated and may warrant premedication with antiemetics or antidiarrheals.[ 16] Ensuring adequate hydration and electrolyte levels in patients experiencing nausea, vomiting, diarrhea, or constipation helps to reduce the consequences of these adverse effects. Although therapy discontinuation due to gastrointestinal adverse effects was required in only 5% of patients, grade 3 or 4 events occurred in 21% of patients.[17]