Proteasome Inhibitors – Bortezomib, Carfilzomib



  • Protein complexes inside all eukaryotes and in some bacteria
  • Located in the nucleus and the cytoplasm
  • Main function – to degrade unneeded/damaged proteins by proteolysis
  • Enzymes involved are proteases
  • Part of a major intracellular homeostasis mechanism by which cells regulate the concentration of unwanted proteins (80%)

Structure of 20S or Catalytic core

  • 20s is a hollow cylindrical particle consists of 4 stacked rings – 2 outer rings and 2 inner rings
  • Each outer ring is composed of 7 different α subunits, while inner ring is composed of 7 different β subunits
  • B ring contains multiple proteolytic activities
      • β5 – has chymotrypsin-like activity ( degrades tyrosine or phenylalanine)
      • β1 – has caspase-like activity (degrades glutamate/acidic residues)
      • β2 – has trypsin-like activity (degrades arginine and lysine)
  • These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded.

Structure of 19S or Regulatory complex

  • Controls the entry of substrate into the catalytic core
  • Each 19s particle consists of a base and a lid
  • The 19S regulatory cap complex recognizes multi-ubiquitinated proteins, unfolds them, removes ubiquitin chains, and provides a passageway for unfolded proteins into the core complex
  • The 19S cap is a 20-subunit 700 kDa complex, also referred to as PA700. When combined with a 20S core complex, it yields a 26S proteasome

Classification of Proteasome Inhibitors

  • Class A – Peptide Aldehydes (e.g. MG115, MG132)
  • Class B – Peptide Boronates (Bortezomib, Ixazomib, Delanzomib)
  • Class C – b-Lactones (Lactacystin, Salinosporamide A, Marizomib)
  • Class D – Epoxy-ketones (Epoxomicin, Carfilzomib, Oprozomib)
  • Class E – Vinyl ketones (Syringolin A, Glidobactin A)




Mechanism of Action

  • Reversible inhibitor of the 26S proteasome – binds to β5 and β1
  • Inactivation of NF-kB – by stabilization of IkB and preventing the translocation of NF-kB to the nucleus
  • Induction of ERS-mediated apoptosis in tumor cells
  • Cell cycle arrest by blocking the degradation of Cyclin B and p27

Mechanisms of resistance

  • Activation of NF-kB pathway via proteasome-independent mechanisms
  • Mutations in the proteasome b5 subunit (PSMB5) gene
  • Increased level of aggresome formation
  • Increased expression of the MDR gene with elevated P170 protein levels – increased drug efflux and decreased intracellular drug accumulation


Absorption – given by the intravenous and SC route


      • VOD – not well characterized
      • plasma protein binding – 80%


      • T ½ – 72-108 hrs
      • metabolized by liver CYP450 – inactive deboronated metabolites
      • elimination pathways – still not characterized


Clinical Trials of Bortezomib


(Phase II data)

Patients Treatment arms Results
Study of Uncontrolled

Myeloma Management with proteasome Inhibition Therapy


Richardson et al. NEJM 2003


patients with relapsed and refractory myeloma

bortezomib 1.3 mg/m2 on Days 1, 4, 8, and 11, for up to 8 cycles.

Dexamethasone 20 mg on the day of and

the day after bortezomib was permitted for suboptimal response

ORR – 35% with bortezomib and 50% with addition of dexa

Responses were independent of the type or number of previous tx, β2-microglobulin level and 13q deletion status

CREST study

Jagannath et al. BJH 2004

54 patients with relapsed myeloma, following 1 line of therapy bortezomib at either 1.0 mg/m2 or 1.3 mg/m2, for maximum 8 cycles, with or without dexamathasone ORR were 33% and 50% with Velcade, and 44% and 62% with addition of dexa

Toxicity – fatigue (70%), PN (41%), thrombocytopenia (30%)

20% less toxicity with low dose Velcade

Assessment of Proteasome inhibition for EXtending

remissions (APEX) trial

Richardson et al. NEJM 2005

randomized phase III trial comparing bortezomib with high dose dexa

in 669 patients with multiple myeloma, relapsed after 1-3 prior therapies

bortezomib 1.3 mg/m2 on Days 1, 4, 8, and 11, for 8 cycles, 3 weekly.

high-dose dexa (40 mg orally) on days 1-4, 9-12, and 17-20 for 4 cycles, 5 weeklY

On progression, pts were allowed to cross over to the Bortezomib arm

ORR – 38% and 18%

CR – 6% vs <1%

median time to progression were 6.2 and 3.4 months

1 yr OS were 80% versus 66%, with bortezomib and dexa, respectively

Velcade as Initial

Standard Therapy in Multiple Myeloma:

Assessment with Melphalan and Prednisone

(VISTA) trial

San Miguel et al. NEJM 2008

Previously untreated 682 pts with myeloma, ineligible for high-dose therapy 9 cycles of melphalan (9 mg/m2) and prednisone (60 mg/m2) on days 1-4, every 6 weekly, either alone

or with bortezomib (1.3 mg/m2) on days 1, 4, 8, 11, 22, 25, 29, and 32 during cycles 1-4 and on days 1, 8, 22, and 29 during cycles 5-9

median time to progression were 24 and 16.6 months

ORR were 71% vs 35%

CR were 30% and 4%

median duration

of the response was 19.9 vs 13.1 months

in the VMP group versus MP, respectively

UPFRONT trial,

Phase III data,

Ruben Niesvizky et al.

JCO 2015

Previously untreated, 502 pts with myeloma, ineligible for transplant 1:1:1 randomised comparison of three frontline Bortezomib-based regimens (VD, VTD, VMP) ORR – 73%, 80% and 70%

Median PFS – 14.7, 15.4 and 17.3 months

Median OS – 49.8, 51.5 and 53.1 months


Phase III data,

Laura Rosinol et al.

Blood 2012

Previously untreated, 386 pts with myeloma, aged <65 yrs, eligible for transplant Randomised comparison of VTD, TD and VBMCP/VBAD/B regimen CR – 35%, 14% and 21 %

Median PFS – 56.2, 28.2 and 35.5 months

Post-ASCT CR – 46%, 24% and 38%

VTD resulted in a higher pre and

Post-transplantation CR rate and in a

significantly longer PFS

Multicenter Phase II Study of Bortezomib in Patients With

Relapsed or Refractory Mantle Cell Lymphoma

Richard I. Fisher et al.

JCO Oct 2006

155 patients of relapsed refractory Mantle cell lymphoma,

Median number of prior therapies was one (range 1-3)

Bortezomib 1.3 mg/m2 was administered on days 1, 4, 8, and 11 of a 21-day cycle, for up to 17 cycles ORR – 33%

CR 8%

Median duration of response – 9.2 mts

TTP – 6.2 months

Toxicity profile –

≥Gr 3 PN – 13%, Fatigue -12%, and thrombocytopenia – 11%

Kieron Dunleavy et al.

Phase II

Blood 2009

49 patients of R/R DLBCL after doxorubicin-based treatment bortezomib

alone followed by bortezomib and


ORR – bortezomib alone -13%, but 83% with chemo + Velcade

median OS – 10.8 vs 3.4 months in ABC compared with GCB DLBCL

Jia Ruan et al.

Phase II

JCO 2011

76 patients with untreated DLBCL (n =40) and MCL (n =36) Received standard R-CHOP 21 with bortezomib (on D 1, 4, 8, 11) for 6 cycles DLBCL – ORR – 100%, CR – 86%, 2-yr OS – 70%

GCB and ABC type – similar results

MCL – ORR – 91%, CR – 72%, 2-yr OS – 86%

Toxicity – PN – 12%

Thrombocytopenia – 25%


Dosage and Strength

  • Recommended dose for relapsed multiple myeloma and mantle cell lymphoma is 1.3 mg/m2 administered by IV twice weekly for 2 weeks (on days 1, 4, 8, and 11) followed by a 10-day rest period (days 12–21) – 3 weekly cycles
  • Reconstitute 3.5 mg vial with 0.9% NaCl
  • IV administration: Add 3.5 mL to vial for final concentration of 1 mg/mL
  • SC administration: Add 1.4 mL to vial for final concentration of 2.5 mg/mL
  • If local injection site reactions occur following SC administration, a less concentrated solution (1 mg/mL) may be administered subcutaneously.


  • Contains no antimicrobial preservative; administer within 8 hr of preparation
  • Not for intrathecal use. Inadvertent intrathecal administration has resulted in death and is contraindicated
  • Separate consecutive doses by at least 72 hours
  • Give IV as a bolus over 3-5 seconds or as SC injection; No central line is required for IV administration.
  • Reconstituted solution does not need to be protected from indoor lighting
  • Do not store reconstituted solution in a syringe for >3 hr
  • Store vial at a controlled room temperature at 25C
  • Each vial of Bortezomib is only for a single use
  • Give SC injection in thigh or abdomen; rotate injection site with each dose

Drug interactions

  • CYP3A4 inhibitors (e.g. azoles, macrolides, protease inhibitors) – increase bortezomib exposure, Ketoconazole increases the exposure by 35%
  • CYP3A4 inducers (e.g. Rifampin, Phenytoin, St. John’s wort) – decrease bortezomib exposure
      • Co-administration of Rifampin, decreases the exposure by 45%
      • Co-administration of dexamethasone, a weak CYP3A4 inducer, had no effect on the exposure

Special precautions

  • Contraindicated in patients with hypersensitivity to boron, bortezomib, and/or mannitol
  • Use with caution in patients with impaired liver function
  • Use with caution in patients with a history of syncope, who are on antihypertensive medications, and who are dehydrated
  • Patients should avoid taking green tea (epigallocatechin gallate)
  • Pregnancy category D – causes post-implantation loss and decreased number of live fetuses
  • Lactation: excretion in milk unknown, however not recommended.

Dose Modifications

Dose Modifications in Neuropathy

Severity of peripheral neuropathy Dose modification
Grade I (Paresthesias and/or loss of reflexes) without pain or loss of function No action
Grade I with pain or Grade II (interfering with function but not with activities of daily living) Reduce VELCADE to 1 mg/m2
Grade II with pain or Grade III (interfering with activities of daily living) Withhold VELCADE until toxicity resolves. When toxicity resolves, reinitiate VELCADE therapy with a reduced dose of 0.7 mg/m2 and change treatment schedule to once per week
Grade IV (sensory neuropathy which is disabling or motor neuropathy that is life threatening or leads to paralysis) Discontinue VELCADE

Dose Modifications in Hepatic Impairment

Severity of impairment Bilirubin level SGOT (AST) levels Modifications of starting dose
Mild 1- 1.5x ULN Any None
Moderate > 1.5 -3x ULN Any Reduce VELCADE to 0.7 mg/m2 in the 1st cycle. Consider dose escalation to 1 mg/m2 or further dose reduction to 0.5 mg/m2 in subsequent cycles based on patient tolerability
Severe > 3x ULN Any

Dose modifications according to hematologic toxicity, when given in combination with Melphalan and Prednisone

Prior to initiating any cycle of therapy –

Platelet count should be at least 70 x 109/L and the absolute neutrophil count (ANC) should be at least 1.0 x 109/L

Toxicity Dose modification or delay
If platelet count is not above 30 × 109/L or ANC is not above 0.75 x 109/L on a

VELCADE dosing day (other than day 1)

Withhold VELCADE dose
If several VELCADE doses in consecutive cycles are withheld due to toxicity Reduce VELCADE dose by 1 dose level (from 1.3 mg/m2 to 1 mg/m2, or from

1 mg/m2 to 0.7 mg/m2)


  • GI toxicity – diarrhea (40-55%), constipation (25%), minimal emetogenic
  • Myelosuppression – thrombocytopenia (30-40%) and neutropenia (15-20%)
  • Peripheral sensory neuropathy (rarely mixed sensorimotor neuropathy and autonomic neuropathy) – (IV – 39%, SC -24%)
  • Fatigue, malaise, and generalized weakness – 40-60%
  • Cardiac toxicity – Acute development or exacerbation of congestive heart failure and new onset of decreased LVEF – 5-8% of patients
  • Orthostatic hypotension in up to 12% of patients
  • Pulmonary toxicity (rare) – ARDS, pneumonitis, interstitial pneumonia
  • Posterior Reversible Encephalopathy Syndrome (PRES) – rare (<1%), but if occurs, then discontinue Velcade
  • Fever (38°C) – 25-40% of patients
  • Hepatic toxicity – hepatitis, increases in liver enzymes, hyperbilirubinemia
  • Herpes zoster reactivation

Bortezomib-induced Peripheral neuropathy (BIPN)

  • Specific mechanism – still unknown
  • Probable mechanism –
      • Bortezomib accumulation in the dorsal root ganglia cells
      • ER-stress
      • ROS mediated injury
  • BIPN may be a proteasome inhibitor class effect, producing primarily a small fiber, painful, distal, sensory neuropathy. Rarely sensory-motor and autonomic neuropathy also
  • Incidence of BIPN
      • Bortezomib pretreated patients – Grade 1/2 – 75%, Grade ≥3 – 30%
      • Bortezomib naïve patients – Grade 1/2 – 30-35%, Grade ≥3 – 15-18%
  • Risk factors – cumulative dose and evidence of pre-existing neuropathy
  • Treatment
      • dose reduction
      • schedule and/or formulation change algorithm (e.g. from twice weekly to once a week and from IV to SC)
      • analgesics, tricyclic antidepressants, anticonvulsants, and vitamin
      • supplements – some symptomatic benefit

Herpes Zoster Reactivation

  • Cell-mediated immunity (CMI) is believed to play a larger role than humoral immunity in prevention of herpes viral reactivation
  • MM is associated with defects in humoral immunity – patients with MM are not at increased risk for herpes zoster infections at baseline
  • Mechanism of herpes zoster reactivation
      • Bortezomib has been shown to interfere with CMI –
      • Inhibitory effects on T-cell proliferation
      • Decreased Th1 cytokines
      • Modulating TLR4-induced dendritic cell activation
  • Dose of Antiviral prophylaxis
      • Acyclovir 400-800 mg BD, Valacyclovir 500 mg BD or Famciclovir 250 mg BD
      • Recent data – a much lower dose of antiviral prophylaxis is equally effective – e.g. Valacyclovir 500 mg OD, Acyclovir 400 mg OD or even 200 mg OD




Mechanisms of action

  • Tetrapeptide epoxyketone inhibitor of the 26S proteasome – irreversibly binds to the to β5 subunit of 20S
  • Down-regulation of the NF-kB pathway
  • Can overcome Bortezomib resistance

Mechanism of resistance

  • Increased expression of MDR gene with elevated P170 protein – increased drug efflux and decreased intracellular accumulation

Advantages of 2nd generation Proteasome inhibitors like Carfilzomib

  • Ease of administration (oral agents such as oprozomib, ixazomib, marizomib, and delanzomib)
  • Abrogating bortezomib-resistant disease with stronger or irreversible binding to the proteasome (carfilzomib, oprozomib, and marizomib
  • Nonpeptide PI (marizomib) is inherently resistant to degradation by endogenous plasma and cellular peptidases resulting in increased bioavailability
  • Have demonstrated single-agent efficacy in refractory disease
  • Have potentially less toxicity


  • Absorption – given only via iv route
  • Distribution
      • VOD not well characterize
      • 97% of the drug bound to plasma proteins
  • Metabolism
      • Rapidly and extensively metabolized by peptidase and epoxide hydrolysis
      • Metabolites have no anti-proteasome activity
      • Liver CYP450 – minor role in metabolism
      • Elimination pathways – not well characterized
      • T ½ – ≤20 hrs


  • FDA-approved (July 2012) for the treatment of patients with multiple myeloma who have received at least 2 prior therapies, including bortezomib and an immunomodulatory therapy

Clinical Trials with Carfilzomib

Source Patients Treatment arms Results
PX-171-003-A1 Phase II study

David S. Siegel et al.

Blood 2012

266 patients with relapsed refractory MM

80% were refractory or intolerant to both bortezomib and lenalidomide.

Patients had median of 5 prior lines of therapy, including bortezomib,

lenalidomide, and thalidomide

78% of pts had Gr I/II PN at the time of entry

single-agent carfilzomib

20 mg/m2 iv twice weekly for 3 of 4 weeks in cycle 1, then 27 mg/m2 for ≤ 12 cycles.

ORR – 23.7%

Median DOR – 7.8 mts

Median OS – 15.6 mts


Fatigue – 49%

Anemia – 46%

Thrombocytopenia – 39%

New onset PN –

12.4% (Gr I, II)

<1% Gr III


Phase II study

Ravi Vij et al.

Blood 2012

129 bortezomib-naive patients with R/R MM (median of 2 prior therapies) Cohort 1, scheduled to receive iv carfilzomib 20 mg/m2 for all treatment

cycles, and Cohort 2, scheduled to receive 20 mg/m2 for cycle 1 and then

27 mg/m2 for all subsequent cycles

Median no of cycles – 7

Cohort 1 had 37% Bortezomib-treated pts

70% of pts had Gr I/II PN at the time of entry

ORR – 42.4 and 52.2% in cohort 1 and 2 respectively

ORR in Bortezomib treated pts of Cohort 1 – 18%


Fatigue – 62%

Nausea – 48%

Thrombocytopenia Gr III – 13%

New onset PN –

17.1% (Gr I, II)

Cohort 1 – 15.3%

Cohort 2 – 18.6%

<1% Gr III PN


  • Recommended dose for cycle 1 is 20 mg/m2/day and if tolerated, the dose can be increased to 27 mg/m2 /day for cycle 2 and all subsequent cycles (in case of Carfilzomib + Lenalidomide + Dexa)
      • can be increased to 56 mg/m2 /day for day 8 of cycle 1 and all
      • subsequent cycles (in case of Carfilzomib + Dexa)
  • Carfilzomib is administered by IV infusion over 10-30 mins, on 2 consecutive days each week for 3 weeks (days 1, 2, 8, 9, 15, and 16) followed by a 12-day rest period (days 17–28)

Preparation and Administration

  • Available in single-use vial (60 mg/vial) – no antimicrobial preservatives
  • Reconstitute each vial with 29 mL Sterile Water for Injection, directing the solution onto the inside wall of the vial to minimize foaming
  • Gently swirl and/or invert the vial slowly for about 1 minute, or until complete dissolution. DO NOT SHAKE to avoid foam generation
  • The reconstituted product should be a clear, colour-less solution
  • Resulting concentration of reconstituted vial is 2 mg/mL


  • Unopened vial containing lyophilized powder: Store refrigerated: between 2-8°C (36-46°F)
  • Reconstituted solution: Stable when refrigerated up to 24 hr (2-8°C; 36-46°F) or at room temperature up to 4 hr

Special precautions

  • Closely monitor for cardiac complications – Patients with prior history of MI in the preceding 6 months, CHF, and conduction system abnormalities not controlled by medication
  • Closely monitor pulmonary status given the risk of pulmonary complications
  • Monitor for infusion-related events – up to 24 hours of administration Premedication with dexa 4 mg PO or IV, at least 30 minutes but no more than 4 hours prior to all doses of cycle 1 and subsequent cycle of dose-escalation. Reinitiate dexa premedication if symptoms occur during other cycles
  • Patients should be well hydrated prior to drug administration to reduce the risk of renal toxicity. The recommended hydration includes both oral fluids (30 ml/kg at least 48 hours before Day 1) and IV fluids (250-500 ml, prior to each dose)
  • Monitor serum electrolytes (K+, Mg2+) regularly during therapy
  • Monitor for TLS
  • Monitor CBC (Thrombocytopenia nadir at day 8), LFT, RFT routinely
  • Pregnancy category D

Dose Modifications

Dose modification according to hemato-toxicity

     Hematologic Toxicity                  Recommended Action            
ANC < 0.5 × 109/L Withhold dose

Upon recovery, start at the same dose level

For subsequent drops of <500/cmm, consider 1 dose level reduction (20 mg/m2)

Platelet count < 10 × 109/L or evidence of bleeding with thrombocytopenia Withhold dose

Upon recovery, start at the same dose level

For subsequent drops of < 10 × 109/L , consider 1 dose level reduction (20 mg/m2)


Dose modification according to renal toxicity

Renal toxicity Recommended action
Serum creatinine > 2 x baseline

Creatinine clearance < 15 ml/min

Creatinine clearance decreases to < 50% of baseline

Need for dialysis

Withhold dose and continue monitoring renal function

Resume when renal function has recovered to within 25% of baseline, start at 1 dose level reduction

For patients on dialysis, the dose is to be given after the dialysis procedure


  • Fatigue, anorexia, and generalized weakness
  • Cardiac toxicity with CHF, myocardial ischemia, and rare cases of cardiac arrest
  • Hypertension – 8% patients
  • Myelosuppression – thrombocytopenia (36%), neutropenia (20%), and anemia
  • Pulmonary toxicity presenting as dyspnea in up to 35% of patients
  • Hepatotoxicity – elevation of transaminases
  • Fever (38°C) is relatively common, seen in up to 40% of patients

Need for novel Proteasome inhibitors

  • Bortezomib therapy in MM is limited by development of peripheral neuropathy and eventual drug resistance mediated via
      • overexpression of the β5 subunit,
      • mutation of active drug binding sites
      • NF-KB activation via non-canonical pathway
  • This mandates the development of novel agents that
  • retain activity in a multi-agent refractory setting
  • are easy to administer (oral vs intravenous)
  • have better tolerance

Second Generation Proteasome Inhibitors

Drug Chemical nature Binding Route
epoxyketone Irreversible IV
(ONX 0912)
 epoxyketone Irreversible ORAL
Ixazomib citrate
 boronate Reversible IV/ORAL
b-lactone Irreversible ORAL
boronate Reversible ORAL


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