The proteasome inhibitor bortezomib (Velcade, PS-341) is prescribed for the treatment of multiple myeloma and mantle cell lymphoma. The second proteasome inhibitor, carfilzomib (Kyprolis, PR-171) 
, was recently approved by the FDA for the treatment of relapsing and refractory myeloma. At least four novel proteasome inhibitors — marizomib (salinosporamide A, NPI-0052) 
, CEP-18770 
, MLN-9708 
, and PR-047 
—are at different stages of clinical development.
Bortezomib is highly cytotoxic to all multiple myeloma cell lines in vitro 
, but in vivo only ~40% of myeloma patients respond to this drug given as a single agent 
. One reason for this discrepancy may be that in vitro concentrations of bortezomib and length of exposure to this agent exceed those that can be achieved in vivo at the maximal tolerated dose (MTD). Most studies of bortezomib in cell culture have utilized continuous incubation for 24–48 h. In the clinical setting, patients receive intravenous or subcutaneous bolus injections twice weekly. When bortezomib is injected intravenously at the MTD, the blood plasma concentration peaks at 100–200 ng/mL (260–520 nM) 5 minutes after IV injection followed by rapid decrease 
. Subcutaneous injection results in ~10-fold lower maximal concentration, which is achieved 30 minutes after injection. The maximal concentration of the drug is maintained for 1–2 h so that total exposure to the drug (area under the Drug concentration-Time curve) is the same as after iv administration. Efficacy of the agent does not depend on the administration route 
The primary target of botezomib is the chymotrypsin-like activity of the proteasome. At the MTD, the mean maximal inhibition of proteasome in patient's peripheral blood cells is 73% after the first dose and up to 83% after subsequent doses 
. This inhibition is achieved within 5–30 min of administration. The inhibition stays at this level for ~1 h and then slowly decreases to 0–25% 48–72 hours later 
. When bortezomib is administered subcutaneously maximal proteasome inhibition in blood is 5% lower than after IV dose and is achieved 2 h after administration. However, the rate of recovery is slightly slower and the area under the effect-time curve is the same as after intravenous administration 
Proteasome inhibition inside MM tumors in patients has not been studied. In a few clinical cases analyzed, inhibition of proteasome in solid tumor biopsies was found to be the same as in blood 
; however, inhibition in bone marrow was found to be half of the inhibition in blood of the same patient 
. Proteasome inhibition in xenograft tumors in mice was ~1/2 of the inhibition in blood 
. Hence we should assume that inhibition of the proteasome inside myeloma tumors does not exceed and most likely is even lower than in blood.
The proteasome core has three pairs of active sites for proteolysis – chymotrypsin-like (ß5), trypsin-like (ß2), and caspase-like (ß1). Cells and tissues of the immune system, including multiple myeloma cells, also contain immunoproteasomes, which express different active-site subunits, i.e., ß5i, ß2i, and ß1i. Chymotrypsin-like sites (ß5, ß5i) are the primary targets of bortezomib, but ß1, ß1i, and ß2i are also inhibited, albeit with lesser potency, and ß2 is activated 
. Inhibition of the proteasome in blood of patients is evaluated based on cumulative inhibition of ß5 and ß5i (chymotrypsin-like) sites.
Although the chymotrypsin-like sites are the most important in protein degradation, our earlier work has indicated that specific inhibition of these sites is not sufficient to block protein breakdown, and co-inhibition of either caspase-like or trypsin-like sites is required 
. This raises the question of how much protein degradation is blocked at clinically achievable levels of proteasome inhibition, when chymotrypsin-likes sites are inhibited by not more than 85%. Inhibition of caspase-like sites and trypsin-like sites has not been reported in clinical samples; but, based on data in multiple myeloma cell lines, we predict that, at the MTD of bortezomib, caspase-like activity is partially inhibited and trypsin-like sites may even be activated. Effects of bortezomib on protein degradation in multiple myeloma cells have not been reported in the literature.
It has been suggested that myeloma cells are particularly sensitive to proteasome inhibitors due to their high ratio of proteasome workload to proteasome expression levels (the “load/capacity” hypothesis) 
. Myeloma cells produce large amounts of immunoglobulins, complex four-chain molecules with several inter- and intra-chain disulfide bridges. Polypeptide chains that fail to fold or assemble have to be degraded via the endoplasmic reticulum (ER)-degradation pathway, imposing a heavy load on the proteasomes of these cells. Consistent with this hypothesis, increased production of immunoglobulins sensitizes myeloma cells to proteasome inhibitors 
In this study, we have compared the effects of continuous bortezomib treatment with a 1-h pulse on a panel of multiple myeloma cell lines. We observed that this shorter incubation increased the differences in sensitivity between cell lines. The most sensitive cell lines underwent apoptosis at clinically achievable bortezomib concentrations and at faster rates than the least sensitive ones; the least sensitive lines remained 100% viable under these conditions. We then found that similar inhibition of active sites by bortezomib causes stronger inhibition of protein breakdown in sensitive cell lines, potentially explaining their enhanced sensitivity.