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Biochemical Pharmacology 96 (2015) 1–9

Contents lists available at ScienceDirect

Biochemical Pharmacology

jo urnal homepage: www.elsevier.com/locate/biochempharm

Commentary

Proteasome inhibitors

Beverly A. Teicher *, Joseph E. Tomaszewski

Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, United States

A R T I C L E I N F O A B S T R A C T

Article history: Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor,

Received 25 February 2015

bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through

Accepted 16 April 2015

development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid

Available online 29 April 2015

inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors

include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and

Keywords:

ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome

Proteasome

inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late

Bortezomib

stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome

Carfilzomib

inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early

Ixazomib citrate

MLN9708 clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more

sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the

proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and

scientific investigation. These agents are continuing to have a major impact in their treatment of

hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer

indications.

Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://

creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction proteasome [2–4]. Timed degradation/recycling of proteins is

essential for viability. The proteasome catabolizes intracellular

The incorporation of the first proteasome inhibitor, bortezomib, proteins thus controlling the intracellular proteins levels to

into anti-myeloma armamentarium can be considered a major maintain cellular function [5–7]. Malignant cells are more

milestone in treatment of multiple myeloma, greatly improving dependent upon the proteasome to remove mis-folded or damaged

the response rates and overall survival in front-line and relapsed/ proteins due to their genetic instability and rapid proliferation.

refractory settings. Although present in all cells, proteasomes are Accumulation of ubiquitinated proteins and/or mis-folded can

relatively abundant in multiple myeloma cells making that disease proteins lead to apoptosis. Proteins involved in cell division,

a target for proteasome inhibitors. proliferation and apoptosis are substrates for the ubiquitin–

proteasome pathway as are mis-folded and mutated proteins.

1.1. The proteasome Proteasomes are abundant and are located in the cytoplasm and

nuclei and of cells. The proteasome refers to the 26S proteasome

Protein activity regulation by synthesis and degradation and includes a 20S core catalytic complex with 19S regulatory

maintains cellular metabolic integrity and proliferation. The subunits on each end (Fig. 1). Proteasomes enter the nucleus

proteasome, a multimeric protease complex, is central to cellular through pores or post mitosis during development of the nuclear

protein regulation by degrading many proteins, thus activating envelope. Proteasome-mediated degradation proteins is enabled

some pathways and shutting down others [1]. Proteins conjugated by conjugation to multiple ubiquitin units. Ubiquitin is a 76-amino

to multiple units of the polypeptide ubiquitin are degraded by the acid protein. Ubiquitin is covalently linked to proteins by an

enzymatic reaction requiring Ub-activating (E1), Ub-conjugating

(E2) and Ub-ligating (E3) enzymes acting sequentially [7]. More

* Corresponding author at: DCTD, National Cancer Institute, RM 4-W602, MSC than four ubiquitin units must be linked to the target protein for

9735, 9609 Medical Center Drive, Bethesda, MD 20892, United States.

proteasome degradation [8]. The S19 regulatory particle guides

Tel.: +1 240 276 5972; fax: +1 240 276 7895.

ubiquitinated proteins into the 20S particle. The 20S core complex

E-mail addresses: [email protected], [email protected]

houses the proteolytic chamber. The 20S complex includes four

(B.A. Teicher).

http://dx.doi.org/10.1016/j.bcp.2015.04.008

0006-2952/Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

2 B.A. Teicher, J.E. Tomaszewski / Biochemical Pharmacology 96 (2015) 1–9

Fig. 1. Illustration of the structure and function of the proteasome. Proteins to be degraded are conjugated with multiple units of ubiqutin then fed in to the proteasome

regulatory particle. The proteins are degraded to peptides by three protease functions in the b subunits of the core particle. The products are free ubiqutin units and peptide

fragments.

ring structures and three proteolytic activities in the b rings provide a more biologically relevant characterization of drug

specifically caspase-like (C-L), trypsin-like (T-L) and chymotryp- action. In early clinical trials, blood samples were collected before

sin-like (CT-L) catalytic proteases. The products produced by the therapy, and then 1, 6, and 24 h after each PS-341 dose for

proteasome are 3–22 amino acid peptides and free ubiquitin pharmacodynamic proteasome activity determination. This assay

protein units (Fig. 1) [9]. provided guidance regarding PS-341 doses near the safe limit of

Early in the study of the proteasome as a potential therapeutic proteasome inhibition which was 80% [19,20].

target, synthetic and natural product inhibitors of the proteasome

were identified [2,9]. Widely studied inhibitors included: lacta- 1.2. Multiple myeloma genetics

cystin, a streptomyces metabolite, which is metabolized to

lactacystin b-lactone, the active proteasome inhibitor [9–12]; Multiple myeloma is divided into two main types: hyperdiploid,

peptide aldehydes, such as carbobenzoxyl-leucinyl-leucinyl-leuc- with multiple trisomies of odd-numbered chromosomes, and

inal-H (MG-132); boronic acid peptides [9,13]; and others nonhyperdiploid, with recurrent immunoglobulin heavy chain gene

[13,14]. Dipeptide boronic acid derivatives, potent proteasome translocations. Multiple myeloma progression is associated with

inhibitors, could be administered to mice, and some were orally MYC rearrangements, the most common multiple myeloma

bioavailable and relatively stable under physiological conditions mutation, present in about 50% of patients. Multiple myeloma

[9,15]. Boronic acid peptides were known to inhibit serine presents with marked subclonal heterogeneity that poses a major

proteases [16–18], whereas the dipeptide boronates had a high challenge to successful disease control. In 1996, Bergsagel et al. [21]

degree of selectivity for the proteasome and were not potent described IGH translocations involving seven partners with break-

inhibitors of many common proteases. points in three B-cell-specific DNA modification processes occur at

Critical to the clinical development of proteasome inhibitors early stages of multiple myeloma pathogenesis [21]. By contrast,

was establishing a pharmacodynamic assay which measured the MYC:IGH rearrangements, generally do not have these IGH break-

potency and duration of inhibition of the proteasomes in normal points in the three B-cell-specific DNA modification processes that

blood cells. In animal-model studies, the dipeptide boronic acid, are inactivated in plasma cells or plasma cell tumors. In multiple

PS-341, rapidly disappeared from the vascular compartment myeloma MYC rearrangements, often combinations of transloca-

[19]. An accurate pharmacodynamic assay was developed to tions, insertions, deletions and inversions, were thought to be late

supplement pharmacokinetic measurements. Both the chymo- events, not involving immunoglobulin genes. However, MYC over-

tryptic and tryptic activities of the proteasome could be detected expression can cause progression to multiple myeloma in a

using fluorogenic kinetic assays. These fluorogenic kinetic assays monoclonal gammopathy of undetermined significance (MGUS)-

were optimized for both whole blood and blood cells. A method for prone mouse strain, as a model for human multiple myeloma.

calculating percent inhibition from the ratio of the enzymatic Multiple myeloma genetic rearrangements can reposition MYC near

activities and knowledge of the catalytic mechanism of the a number of conventional enhancers, and often genes with super-

proteasome was developed using two structurally unrelated enhancers. Multiple myeloma lacking MYC rearrangement have

inhibitors (PS-341 and lactacystin). The 20S proteasome concen- MYC expression at higher levels than in MGUS. However, germinal

tration in whole blood and red blood cells was equivalent, thus, center MYC activation did not cause multiple myeloma in a mouse

data collected from either source provided similar proteasome strain that rarely develops MGUS. MYC rearrangements contribute

inhibition. Cellular response to PS-341 was shown to be due to the to multiple myeloma autonomy [22].

drug availability rather than cellular proteasome concentration. Massively parallel sequencing of paired tumor/normal samples

PS-341 rapidly disappeared from the intravascular compartment from 203 multiple myeloma specimens identified mutated genes,

and distributed widely. Serum concentrations approached the copy number alterations and putative tumor suppressor genes

limit of pharmacokinetic detection within 1 h. The pharmacoki- based on homozygous deletions and loss of heterozygosity. KRAS

netic measurements were not sensitive enough to guide dosing for mutations were seen frequently in previously treated patients, as

PS-341, however, the pharmacodynamic assay measuring the were NRAS, BRAF, FAM46C, TP53, and DIS3 mutations. Mutations

percentage of inhibition of proteasome function could be used to were present in subclonal populations, and multiple mutations in

B.A. Teicher, J.E. Tomaszewski / Biochemical Pharmacology 96 (2015) 1–9 3

the same pathway were frequent [23]. Hyperdiploid and non- proteasome inhibitors to treat muscle-wasting conditions. Julian

hyperdiploid changes appear to be early or even initiating Adams, a medicinal chemist, was recruited to head the company

mutagenic events that are followed by additional aberrations [3]. Since proteasome inhibitors were quite toxic, a new

including copy number abnormalities, translocations, mutations, therapeutic focus was needed for commercialization. The lead

and epigenetic modifications leading to plasma cell immortaliza- compound, MG-341, selectively inhibited the chymotryptic

tion and disease progression [24]. activity of the 20S proteasome (Fig. 2). In 1994, the activity of

the boronic acid dipeptide proteasome inhibitor MG-341, was

examined cancer cell lines and mouse tumor models at Dana

2. Bortezomib

Farber Cancer Institute. Publication of the results was held up for

In 1993, Alfred Goldberg, the discoverer of the proteasome, several years during which time MyoGenetics became ProScript

and colleagues founded the company MyoGenetics to develop and MG-341 became PS-341 (Fig. 3 and Table 1). PS-341 was a

Fig. 2. Timeline for proteasome inhibitor development. Bortezomib (PS-341) Ixazomib citrate (MLN9708) Ixazomib (MLN2238)

NSC681239 NSC758254 NSC766907 Carfilzomib (PR171) Delanzomib (CEP18770) Oprozomib (ONX0912) NSC758252 NSC764334 NSC766254

Marizomib (NPI-0052)

Fig. 3. Chemical structures of proteasome inhibitors.

4 B.A. Teicher, J.E. Tomaszewski / Biochemical Pharmacology 96 (2015) 1–9

Table 1

Characteristics of proteasome inhibitors including chemical class, potency in inhibiting proteasome protease activities, and clinical circulating half-life and major route of

administration.

Active moiety Binding CT-L IC50 C-L IC50 T-L IC50 Circulating Route Refs.

(nM) (nM) (nM) half-life (min)

Bortezomib (PS-341) Boronate Reversible 7.9 Æ 0.5 53 Æ 10 590 Æ 67 110 Intravenous [59]

Carfilzomib (PR-171) Epoxyketone Irreversible <5 2400 3600 <30 Intravenous [60]

Marizomib (NPI-0052) b-Lactone Irreversible 3.5 Æ 0.3 430 Æ 34 28 Æ 2 10–15 Intravenous [61]

Ixazomib (MLN9708) Boronate Reversible 3.4 31 3500 18 Oral [59,61]

Oprozomib (ONX0912) Epoxyketone Irreversible 36–82 ND ND 30–90 Oral [63,64]

Delanzomib (CEP-18770) Boronate Reversible 3.8 ND ND ND Oral [51]

potent cytotoxic agent in MCF-7 human breast carcinoma cells, molecular consequences of PS-341 exposure in multiple myeloma

producing an IC90 of 0.05 mM after 24 h exposure. PS-341 was cells showed down-regulation of growth/survival signaling path-

potently cytotoxic when administered to mice bearing EMT-6 ways, and up-regulation of molecules involved in pro-apoptotic

tumors. PS-341 was also toxic to bone marrow CFU-GM from the pathways consistent with proteasome inhibition, as well as up-

femurs of the same mice. PS-341 increased the tumor cell killing of regulation of heat-shock proteins and ubiquitin/proteasome

radiation therapy, cyclophosphamide, and cisplatin in mice pathway proteins consistent with stress responses [28]. Sub-

bearing EMT-6 tumors. In tumor growth delay assays, PS-341 cytotoxic PS-341 concentrations sensitized multiple myeloma

had antitumor activity against primary Lewis lung carcinoma as lines and patient cells to DNA-damaging chemotherapeutic drugs

well as against spontaneous lung metastases in the same mice. In including doxorubicin and melphalan. Sensitization was observed

combination, with 5-fluorouracil, cisplatin, paclitaxel and doxoru- in cells resistant to DNA-damaging drugs and cells isolated from a

bicin, PS-341 produced additive tumor growth delays against patient who relapsed after PS-341 monotherapy. PS-341 complete-

subcutaneous primary tumors and was highly effective against ly blocked cell adhesion-mediated drug resistance. As determined

disease metastatic to the lungs [6]. by gene expression profiling and proteomic analysis, PS-341 down-

In 1995, PS-341 (NSC681239) along with several analogs (NSCs: regulated the expression of effectors involved in cellular response

681226, 681228, 681229, 681231, 681234, 68236, and 681237), to genotoxic stress. The data indicate that PS-341 down-regulated

was submitted to the NCI-60 cell line screen and was found to have apoptosis inhibitors expression, and inhibited genotoxic stress

potent, wide-ranging activity in solid and liquid tumor cell lines response pathways, thus, increasing sensitivity to DNA-damaging

(http://dtp.nci.nih.gov). There was good correlation between chemotherapeutic drugs [29].

proteasome inhibition and cell line response among tested ProScript was acquired by Millennium Pharmaceuticals in 1999

compounds. PS-341 produced a unique pattern in the COMPARE (Fig. 2). Critical to enabling the clinical development of PS-341 was

analysis, suggesting that PS-341 had a novel mechanism of action. the creation of a suitable clinical formulation of stable composition

PS-341 had antitumor activity in several human solid tumor that released the boronic acid compound in aqueous media

xenografts [25]. [30]. The clinical formulation and preclinical safety testing of PS-

In 1996, nuclear factor kB (NF-kB) activity was recognized as a 341 with this formulation was carried out by the Developmental

pro-tumor cell survival protein in multiple myeloma. Normally, Therapeutics Program of the National Cancer Institute. Several

the function of NF-kB is inhibited by binding to the inhibitor, IkB. Phase 1 clinical trials were conducted with bortezomib varying the

Release of activated NF-kB occurs after proteasome-mediated dosing schedule and patient groups. A breakthrough Phase

degradation of ubiqutin-conjugated IkB. Thus, PS-341 indirectly 1 clinical trial determined the maximum-tolerated dose (MTD),

inhibited activation of NF-kB. PS-341 was cytotoxic toward four dose-limiting toxicity (DLT), and pharmacodynamics of bortezo-

murine and two human squamous cell carcinoma cell lines via mib in patients with refractory hematologic malignancies

apoptosis. PS-341 inhibited growth of murine and human [32]. Patients were treated with PS-341 2-times per week for

2

squamous cell carcinoma tumors in mice at doses of 1–2 mg/kg 4 weeks at doses ranging from 0.40 to 1.38 mg/m followed by a 2-

2

(3–6 mg/m ) given three times weekly [25]. Multiple myeloma week rest. PS-341 pharmacodynamics was evaluated by measure-

cells have increased NF-kB activity compared with normal ment of whole blood 20S proteasome activity [19,31]. Twenty-

hematopoietic cells. PS-341 blocked nuclear translocation of NF- seven patients received 293 PS-341 doses comprising 24 complete

kB, and demonstrated antitumor activity against chemo-resistant cycles. Dose limiting toxicities occurred at doses greater than

2

and chemo-sensitive multiple myeloma cells alone and in models 1.04 mg/m included fatigue, thrombocytopenia, hyponatremia,

of the bone marrow microenvironment both in vitro and in vivo hypokalemia, and malaise. PS-341 caused 20S proteasome

multiple myeloma xenografts. The sensitivity of chemo-resistant inhibition in a time-dependent manner, and percent proteasome

myeloma cells to chemotherapeutic agents was increased when inhibition was related to PS-341 dose. Among the 9 assessable

combined with PS-341 [4]. Ma et al. [26] showed that PS-341 patients with heavily pretreated plasma cell malignancies

inhibited proliferation and induced cell death in human multiple completing one cycle of therapy, there was one complete response

myeloma cell lines and freshly isolated multiple myeloma clinical (CR) and a reduction in paraprotein and/or marrow plasmacytosis

samples; inhibited MAP kinase signaling in multiple myeloma in 8 others. A mantle cell lymphoma patient and a follicular

cells; caused apoptosis in p53 wild-type and p53 mutant multiple lymphoma patient had shrinkage of nodal disease. PS-341 was

2

myeloma cells; overcame drug resistance; increased dexametha- well-tolerated at 1.04 mg/m on the twice weekly i.v. bolus for

sone anti-multiple myeloma activity; and overcame the protection 2 weeks, followed by a 1-week recovery period schedule. Thus, PS-

from apoptosis in multiple myeloma cells by interleukin-6. PS-341 341 was clinically active against refractory multiple myeloma and

inhibited multiple myeloma cells growth by decreasing adherence possibly non-Hodgkin’s lymphoma [32]. In 2002, a second Phase

to bone marrow stromal cells and NF-kB-dependent interleukin-6 1 clinical trial evaluated the toxicity and pharmacodynamic

production. PS-341 acted both on multiple myeloma cells and on behavior of PS-341 administered 2-times per week by intravenous

cellular interactions and cytokine secretion in the bone marrow bolus injection for 2 weeks, followed by a 1 week recovery period

milieu to block tumor cell growth, cause apoptosis, and overcome in patients with advanced solid tumor malignancies [31]. In this

drug resistance [27]. Gene expression profiling indicated that the Phase 1 trial, 43 patients were treated with PS-341 at doses ranging

B.A. Teicher, J.E. Tomaszewski / Biochemical Pharmacology 96 (2015) 1–9 5

2

from 0.13 to 1.56 mg/m /dose for a total of 89 cycles. Pharmaco- 3.5 months in the dexamethasone arm. The overall response rate

dynamic studies were performed to determine 20S proteasome (complete responses plus partial responses) with bortezomib was

activity. PS-341 dose-limiting toxicities in this trial were diarrhea 38% compared with a dexamethasone overall response rate of 18%.

and sensory neurotoxicity. There was no dose-limiting hemato- On June 23, 2008, the U.S. FDA approved bortezomib for the

logical toxicity. A PS-341 dose related inhibition of 20S proteasome treatment of patients with multiple myeloma as a first-line

activity was documented. A patient with refractory non-small cell therapy for patients. In 2006, bortezomib was approved for

lung carcinoma had a one major clinical response. The dose of treatment of relapsed or refractory mantle cell lymphoma, and in

2

1.04 mg/m /dose administered twice weekly for 4 weeks followed 2014, bortezomib was approved for first-line therapy of patients

by a 2-week rest, was recommended for Phase 2 trials [31]. with mantle cell lymphoma.

These Phase 1 clinical trials, along with preclinical findings of Bortezomib was the first proteasome inhibitor approved for the

anti-myeloma activity, provided the impetus for a multicenter, therapy of multiple myeloma. Although bortezomib was a great

open-label, non-randomized Phase 2 bortezomib clinical trial for step forward in the multiple myeloma treatment, a sub-population

relapsed, refractory multiple myeloma treatment. The Phase 2 trial of patients failed to respond to bortezomib and almost all patients

reported in 2003 enrolled 202 patients. Patients received 1.3 mg of relapsed after brotezomib either as a single agent or when used in

2

bortezomib/m 2-times per week for 2 weeks, followed by 1 week combination. The positive clinical outcome of bortezomib treat-

without treatment, for up to 8 cycles. In some patients, oral ment in multiple myeloma patients provided impetus for the

dexamethasone (20 mg) was added to the regimen on the day of discovery and development of next generation proteasome

and the day after bortezomib administration. Response was inhibitors with improved efficacy and safety [35,36].

evaluated according to the European Group for Blood and Marrow Despite the great success of bortezomib, there are some

Transplantation criteria and by an independent review committee. patients who do not respond to the drug or respond briefly, then

Of 193 evaluable patients, 92% were previously treated with three relapse. Bortezomib reversibly inhibits the CT-like activity in the

or more agents for myeloma, and 91% of patients were refractory to proteasome b core particle. Mutations and/or over-expression of

the most recent therapy. The response rate to bortezomib was b subunits is a frequent cause of bortezomib resistance. Point

35%, including 7 patients in whom myeloma protein became mutations in PSMB5 of the b subunits are an important

undetectable and 12 patients with myeloma protein detectable mechanism of bortezomib resistance. Clonal sub-lines of the

only by immunofixation. The median overall survival was HT-29 adenocarcinoma line made resistance to bortezomib in cell

16 months, and the median duration of response was 12 months. culture were 30-fold resistant to bortezomib. Two novel and

Grade 3 adverse events included thrombocytopenia, fatigue, distinct mutations in the b subunits were identified in the

peripheral neuropathy, and neutropenia [33]. This trial was botrezoimb resistant clones. One of these mutations has been

uncontrolled, however, several processes were in place to reduce found in bortezomib-resistant multiple myeloma patients.

bias in analyzing response to therapy. Each patient was his or her Proteasome activity and levels were increased in some bortezo-

own control in the assessment of time to progression of disease mib-resistance clones. These changes correlated with a more

relative to that with the last therapy received before enrollment, rapid recovery of proteasome activity following exposure to

and an analysis was performed to elucidate an association between bortezomib [37]. Modeling studies suggestion that the docu-

response to bortezomib alone and survival. The median duration of mented mutations decrease bortezomb binding affinity resulting

survival among patients who did not respond was 8 months and in the decreased duration of inhibition of the CT-like activity by

was in the range expected. Response to bortezomib did not bortezomib. Alterations in gene and/or protein expression in

correlate with many standard multiple myeloma prognostic stress, survival, and anti-apoptotic pathways are contributing

factors such as deletion of chromosome 13, a predictor for poor bortezomib resistance mechanisms [38].

outcome with conventional therapy. The time to progression with

bortezomib therapy increased by a factor of 2–4, compared with 3. Carfilzomib

the most recent therapy patients received before entering the trial.

Responses were associated with increased hemoglobin and Bortezomib success stimulated broad investigation of the

decreased transfusions, improved quality of life, and increased proteasome as an anticancer target. Carfilzomib (PR-171) is an

normal immunoglobulins. In 2003, bortezomib (Velcade, PS-341) irreversible epoxyketone proteasome inhibitor. Carfilzomib is

was approved by the U.S. Food and Drug Administration (U.S. FDA) chemically, structurally and mechanistically different from borte-

for the treatment of refractory multiple myeloma. Bortezomib zomib. Carfilzomib is equally potent but more selective for the

slowed progression of myeloma bone disease. This beneficial chymotrypsin-like activity of the proteasome than bortezomib

impact of bortezomib on bone metabolism was not due to anti- (Fig. 3 and Table 1). In cell culture, carfilzomib is more cytotoxic

myeloma activity; bortezomib directly interferes with osteoclas- than bortezomib following brief treatments at the same concen-

togenesis and resorption and promotes osteoblast formation and tration under conditions that mimicked the pharmacokinetics of

function. both compounds. Hematologic tumor cells were most sensitive to

A randomized, multicenter Phase 3 trial comparing bortezo- carfilzomib exposure, compared with solid tumor cells and non-

mib with high-dose dexamethasone in relapsed multiple myelo- transformed cell types. Cellular consequences of carfilzomib

ma patients was initiated [34]. In 2005, bortezomib received exposure included accumulation of proteasome substrates, induc-

regular approval from the U.S. FDA for the treatment of multiple tion of cell cycle arrest, and apoptosis. Carfilzomib administration

myeloma progressing after at least one prior therapy. The to mice resulted in dose-dependent inhibition of the chymotryp-

approval was based on the large, comparative international sin-like proteasome activity in all tissues examined except the

open-label Phase 3 trial that randomized 669 multiple myeloma brain. Carfilzomib was well-tolerated in mice when administered

patients treated with at least one previous systemic regimen to for 2 days at 5 mg/kg or for 5 days at 2 mg/kg These regimens

receive bortezomib or high-dose dexamethasone in which resulted in >80% proteasome inhibition in blood and most tissues.

bortezomib efficacy and safety was clear. At the pre-planned Carfilzomib anticancer activity was both dose and schedule

interim analysis, the independent data-monitoring committee dependent in human tumor xenograft models [39]. Proteasome

recommended that bortezomib be offered to all dexamethasone- inhibition was longer with carfilzomib due to covalent bonding of

treated study patients because the time to disease progression the compound to the target protein, than with bortezomib.

was 6.2 months in the bortezomib treatment arm compared with The carfilzomib epoxyketone pharmacophore is selective for the

6 B.A. Teicher, J.E. Tomaszewski / Biochemical Pharmacology 96 (2015) 1–9

NH2-terminal threonine residue that catalyzes enzymatic activity ed growth inhibition, apoptosis, and decreased angiogenesis.

in each of the proteolytic active sites in the proteasome. Combining MLN2238 with lenalidomide, vorinostat, or dexameth-

In a Phase 1 clinical trial, reported in 2009, assessing carfilzomib asone increased cytotoxicity and anticancer activity [45].

safety and efficacy in patients with relapsed or refractory Ixazomib (MLN9708/MLN2238) was the first oral proteasome

hematologic malignancies, 29 patients were enrolled who after inhibitor to be assessed for the treatment of multiple myeloma in

at least two prior therapies had relapsed or refractory disease. patients. Ixazomib had very different pharmacokinetic and

Carfilzomib antitumor activity was observed at doses 11 mg/ pharmacodynamic parameters than bortezomib, in addition to

2

m . The Phase 1 carfilzomib clinical trial had one patient with similar efficacy in multiple myeloma growth control and bone loss

mantle cell lymphoma had an unconfirmed complete response, prevention. Ixazomib was not cross-resistant with bortezomib.

one patient with multiple myeloma had a partial response, and one Phase 1/2 clinical studies using ixazomib once weekly or twice

patient with multiple myeloma and one patient with Walden- weekly in relapsed/refractory multiple myeloma patients sug-

stro¨m’s macroglobulinemia had minimal responses. Of, thus, gested single agent antitumor activity, however, promising clinical

indicating that carfilzomib was tolerable and had clinical activity results were obtained with the combination of ixazomib,

in multiple hematologic malignancies [40]. In 2012, carfilzomib lenalidomide, and dexamethasone in newly diagnosed multiple

was approved for the treatment of multiple myeloma patients who myeloma patients. Ixazomib has been tested in systemic amyloid-

have received at least two prior therapies including bortezomib osis as a single agent, showing important activity in this difficult

and an immunomodulatory agent [41]. plasma cell dyscrasia [46]. Clinical trials of orally administered

ixazomib (now in Phase 3) indicate that ixazomib is tolerable with

4. Marizomib infrequent peripheral neuropathy and clinical anti-myeloma

activity either alone or in combination with other anti-myeloma

Marizomib (NPI-0052, Salinosporamide A) is a naturally drugs with signs of additional beneficial bone effects [47–49].

occurring g-lactam-b-lactone bicyclic compound which is an In a Phase 1 clinical trial, 60 patients with relapsed/refractory

irreversible proteasome inhibitor. Marizomib inhibits all three multiple myeloma who had a median of 4 prior therapeutic

proteasome protease activities and is not cross-resistant with regimens including bortezomib, lenalidomide, thalidomide, and

bortezomib. In human multiple myeloma xenografts marizomib is carfilzomib and marizomib, received single-agent ixazomib on

an active antitumor agent which is well tolerated, and extends days 1, 4, 8, and 11 in 21-day cycles. The maximum tolerated dose

2

survival [42]. Chemically, marizomib has a leaving group, which was 2.0 mg/m . Patients received a median of 4 cycles with a range

increases cytotoxic potency in RPMI 8226 multiple myeloma and of 1–39. Eighteen percent of patients received 12 cycles. The

PC-3 prostate adenocarcinoma cells. Marizomib effluxes slowly ixazomib terminal half-life was 3.3–7.4 days and the plasma

from cells due to irreversible binding to the proteasome exposure increased proportionally with ixazomib dose. Among the

[43]. Pharmacodynamic assay showed that marizomib is a potent evaluable patients, 15% achieved a partial response and 76%

proteasome inhibitor and that in vivo proteasome inhibition is achieved stable disease [50]. Another Phase 1 trial enrolled

dose, cycle and schedule dependent. Pharmacokinetic assay 60 patients with relapsed, refractory multiple myeloma to evaluate

indicated that marizomib has a short half-life with rapid clearance safety and tolerability and determine the maximum tolerated dose

and a large volume of distribution. Marizomib exposure increases of single agent, oral ixazomib administered weekly for 3 of

linearly with dose. In single agent Phase 1 trials, marizomib 4 weeks. Upon maximum tolerated dose determination, patients

administered on a day 1, 8, and 15 schedule was tolerated up to were enrolled to 4 different cohorts based on clinical status and

2

0.4–0.6 mg/m [36]. In one Phase 1 trial which included 34 patients, prior bortezomib and carfilzomib exposure. The ixazomib maxi-

2

88% had received prior bortezomib, and 71% were refractory to mum tolerated dose was 2.97 mg/m . Nine patients achieved a

2

bortezomib. The marizomib MTD was 0.4 mg/m when adminis- partial response, including 8 evaluable patients treated at the MTD.

2

tered as a 60-minute intravenous infusion and 0.5 mg/m when Pharmacokinetic studies indicated a long terminal half-life for the

administered as a 120-minute intravenous infusion. Three of compound of 3.6–11.3 days [51,52]. Ixazomib is currently in

15 patients treated in the active dose range achieved a PR multiple Phase 3 clinical trials.

[44]. Marizomib is in Phase 1/2 clinical trial in relapsed or

refractory multiple myeloma and grade IV malignant glioma 6. Oprozomib

(clinicaltrials.gov).

The novel proteasome irreversible inhibitor, oprozomib (ONX

5. Ixazomib 0912) is a tripeptide epoxyketone and can be described as an orally

bioavailable analog of carfilzomib (Fig. 3 and Table 1). Oprozomib

Ixazomib (MLN9708/MLN2238), a novel orally bioactive inhibited growth and induced apoptosis in multiple myeloma cells

boronic acid proteasome inhibitor, was tested for activity against resistant to conventional therapy and bortezomib. Oprozomib

in multiple myeloma using well-established in cell culture and in cytotoxicity was simultaneous with activation of caspase-8,

tumor models [45]. Multiple myeloma cell lines, primary patient caspase-9, caspase-3, and poly(ADP) ribose polymerase. Oprozo-

cells, and a human multiple myeloma xenograft tumor model were mib inhibited of migration of multiple myeloma cells. Like

used in the initial study of MLN9708/MLN2238 antitumor activity. bortezomib, oprozomib, inhibited proteasome chymotrypsin-like

MLN2238 inhibited the chymotrypsin-like activity of the protea- activity. In mouse tumor models, oprozomib inhibited tumor

some in multiple myeloma cells and induced accumulation of progression and prolonged survival. Immuno-staining of multiple

multiply ubiquitinated proteins (Fig. 3 and Table 1). MLN2238 myeloma tumors from oprozomib-treated mice showed apoptosis,

inhibited growth and caused apoptosis in multiple myeloma cells and decreased angiogenesis. Finally, oprozomib enhanced the

resistant to conventional cytotoxic compounds and bortezomib activity of bortezomib, lenalidomide, dexamethasone, and a pan-

without affecting the viability of normal cells. In xenograft tumor histone deacetylase inhibitor [53]. Interactions between multiple

models, MLN2238 was well-tolerated and slowed tumor growth. myeloma cells and the bone marrow microenvironment increased

Comparison of MLN2238 and bortezomib in a tumor model the number and activity of bone-resorbing osteoclasts while

showed a longer survival time in mice treated with MLN2238 than decreasing bone-forming osteoblasts resulting in increased tumor

mice treated with bortezomib. Immuno-staining of multiple growth and osteolytic lesions. At clinically relevant concentrations,

myeloma xenograft tumors from MLN2238-treated mice elucidat- carfilzomib and oprozomib inhibited osteoclast formation and

B.A. Teicher, J.E. Tomaszewski / Biochemical Pharmacology 96 (2015) 1–9 7

bone resorption in culture, and enhanced osteogenic differentia- Delanzomib safety, pharmacokinetics and pharmacodynamics

tion and matrix mineralization. Carfilzomib and oprozomib of, were assessed after intravenous administration on days 1, 4,

increased trabecular bone volume, decreased bone resorption 8 and 11 of an every 21 days cycle in patients with solid tumors and

and increased bone formation in non-tumor bearing mice. In multiple myeloma. Thirty-eight patients were treated with

2

mouse bearing disseminated multiple myeloma, carfilzomib and delanzomib at doses ranging from 0.1 to 1.8 mg/m . The recom-

2

oprozomib decreased tumor burden and prevented bone loss mended Phase 2 dose of 1.5 mg/m was tested in additional

[53]. There is an on-going open-label Phase 1/2 study of the patients. Delanzomib pharmacokinetics in plasma was linear with

efficacy and safety of oprozomib hematologic malignancy patients a half-life of the elimination phase of 62.0 Æ 43.5 h. Proteasome

[54]. The objectives of Phase 1b were to determine oprozomib inhibition in peripheral blood mononuclear cells was dose related in

maximum tolerated dose and the safety and tolerability. The multiple myeloma patients [60]. Delanzomib has completed Phase

objective of the Phase 2 portion of the trial was to determine 1 clinical trial (clinicaltrials.gov).

the overall response rate. Oprozomib was administered once daily

on days 1, 2, 8, and 9 of a 14-day cycle or on days 1–5 of a 14-day 8. Other indications

cycle. The starting dose was 150 mg/day and was increased to

330 mg/day. In the Phase 2 portion of the trial, the oprozomib dose There are no U.S. FDA approved treatment for patients affected

was 240 mg/day on days 1–5 of a 14-day cycle. In the Phase by the recessively inherited, progressive muscular dystrophies

2 portion of the trial using the days 1–5 schedule, the overall caused by muscle membrane repair protein dysferlin deficiency.

response rate in carfilzomib-refractory patients and carfilzomib- Marked reduction in dysferlin in patients harboring missense

sensitive patients was 27.3% and the overall response rate among mutations implies that dysferlin is degraded by the cell quality

bortezomib-refractory patients was 25.0% (clinical trials.gov) control machinery. Data suggest that missense mutated dysferlin

[55]. might be functional if salvaged from degradation by the protea-

some. Three patients with muscular dystrophy due to homozygous

7. Delanzomib mutation in dysferlin were treated with the proteasome inhibitor

bortezomib. Dysferlin expression was monitored in monocytes and

Delanzomib (CEP-18770) is an orally bioavailable inhibitor of in skeletal muscle by biopsy. Expression of missense mutated

the proteasome chymotrypsin-like activity that decreases NF-kB dysferlin in the skeletal muscle and monocytes of the three

activity and the expression of several NF-kB downstream effectors patients increased, and dysferlin correctly localized to the

(Fig. 3 and Table 1). Delanzomib caused apoptotic cell death in sarcolemma of muscle fibers. Salvaged missense mutated dysferlin

multiple myeloma cell lines and in purified CD138-positive was functional in patient-derived muscle cells treated with three

primary cells from untreated and bortezomib-treated multiple different proteasome inhibitors. Proteasome inhibition increased

myeloma patients. In culture, delanzomib had anti-angiogenic expression of missense mutated dysferlin, suggesting that this

activity and suppressed RANKL osteoclastogenesis. Delanzomib therapeutic strategy may benefit patients with dysferlinopathies

was less cytotoxic toward normal human epithelial cells, bone and possibly other genetic diseases [61].

marrow progenitors, and bone marrow-derived stromal cells than Late-stage systemic lupus erythematosus (SLE) is treated with

toward multiple myeloma cells. Delanzomib intravenous or oral cytotoxic agents. Delanzomib was tested in comparison with

administration to mice resulted in a sustained pharmacodynamic bortezomib to treat fatal lupus nephritis (LN) in mice. Age matched

inhibition of proteasome activity in tumors relative to normal mice with established SLE or LN, were treated with delanzomib or

tissues, and resulted in complete regression of multiple myeloma with bortezomib. Reduction of specific anti-chromatin, dsDNA

xenografts and in a systemic model of multiple myeloma increased antibody secreting cells and circulating antinuclear antibodies,

median survival [56]. Delanzomib in combination with dexameth- were observed following delanzomib treatment. Reductions in

asone or lenalidomide resulted in longer tumor growth delays pro-inflammatory cytokines were observed in delanzomib-treated

compared to vehicle alone, each drug alone, or the dexamethasone animals. Delanzomib treatment suppressed the development and

and lenalidomide doublet. The three xenograft studies suggested progression of renal tissue damage and extended the survival of ill

that delanzomib in combination with dexamethasone and mice. Proteinuria was decreased and severity of renal histopathol-

lenalidomide may be useful for multiple myeloma treatment ogies reduced relative to vehicle-treated nephritic mice. Delanzo-

[57]. Bortezomib and delanzomib were compared for proteasome mib treatment resulted in improved stabilization of disease [62].

inhibitory activity using fluorogenic substrates and a fluorescent

proteasome activity probe. Bortezomib and delanzomib were 9. Conclusion

equipotent in inhibiting distinct subunits of the proteasome in a

panel of cell lines. In a multiple myeloma model, tumor Proteasome inhibitors are a recent addition to the anticancer

proteasome activity was inhibited to a greater extent by armentarium. While cancer was not the first therapeutic target

delanzomib then by bortezomib. Delanzomib was not cross disease for bortezomib, the potency and toxicity of the agent re-

resistant with bortezomib in vitro [58]. Oral or intravenous focused bortezomib development into cancer. After such a shaky

delanzomib administration was evaluated in mice bearing beginning, bortezomib and a company NCI partnership, became a

multiple myeloma tumors alone and in combination with breakthrough drug for treatment of multiple myeloma, a cancer

conventional therapeutics. Delanzomib combined with melphalan that had not seen a therapeutic improvement for many years. The

or bortezomib resulted in synergistic inhibition of multiple clinical and commercial successful of bortezomib stimulated

myeloma cell viability. In multiple myeloma xenografts, the several firms to search for second-generation proteasome inhibi-

addition of intravenous delanzomib to melphalan prevented the tors with improved therapeutic indices and oral bioavailability

growth of melphalan-sensitive and melphalan-resistant tumors. compared with bortezomib. Bortezomib is a boronic acid requiring

The combination of intravenous delanzomib and bortezomib intravenous administration while ixazomib and delanzomib are

resulted in complete regression of bortezomib-sensitive tumors boronic acids that are taken orally. Carfilzomib is an irreversible

and delayed progression of bortezomib-resistant tumors com- epoxyketone requiring intravenous administration, while oprozo-

pared with either agent alone. Single agent oral delanzomib mib is an epoxyketone that is taken orally. These compounds are

produced anti-multiple myeloma effects in the same xenograft generally very potent cytotoxics, all of which focus on blocking the

models [59]. proteasome chymotrypsin-like protease. The focus on multiple

8 B.A. Teicher, J.E. Tomaszewski / Biochemical Pharmacology 96 (2015) 1–9

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