Novel, orally active, proteasome inhibitor, delanzomib (CEP-18770), ameliorates disease symptoms and glomerulonephritis in two preclinical mouse models of SLE
Matthew M. Seavey ⁎, Lily D. Lu, Kristine L. Stump, Nate H. Wallace, Bruce A. Ruggeri
Cephalon, Inc., Worldwide Discovery Research, 145 Brandywine Parkway, West Chester, PA 19380-4249, United States
a r t i c l e i n f o
Article history:
Received 31 August 2011
Received in revised form 20 October 2011 Accepted 29 November 2011
Available online 13 December 2011
Keywords: Proteasome inhibitor Mouse lupus model Small molecule
a b s t r a c t
Current therapies for late-stage systemic lupus erythematosus (SLE) are limited to cytotoxic agents. Delanzo- mib (CEP-18770) is an orally active, reversible P2 threonine boronic acid inhibitor of the 26S mammalian proteasome. Delanzomib was tested in a head-to-head comparison against bortezomib to protect and treat mice with fatal lupus nephritis (LN). Age matched MRL/lpr or NZBWF1 mice with established SLE or LN, re- spectively, were treated with delanzomib either 3 mg/kg once or twice weekly intravenously or orally at 10 mg/kg. Mice were also treated with reference agent bortezomib at 0.5 mg/kg, intraperitoneally, once a week or 0.3 mg/kg once or twice a week. Reductions in the frequencies of specific anti-chromatin, smith and dsDNA antibody secreting cells and levels of the corresponding circulating antinuclear antibodies, were observed following delanzomib treatment. Reductions in several serum pro-inflammatory cytokines were observed in delanzomib-treated animals. Delanzomib treatment suppressed the development and pro- gression of renal tissue damage and extended the survival of ill mice. Proteinuria was significantly decreased and severity of various renal histopathologies reduced relative to vehicle-treated nephritic mice. Treatment of lupus in these models demonstrated that delanzomib treatment lead to greater tolerability and rate of re- sponse resulting in improved stabilization of disease.
Published by Elsevier B.V.
1. Introduction
The treatment of antibody-driven diseases such as Sjögren’s Syn- drome (SS), Myasthenia Gravis, Hashimoto’s Thyroiditis and systemic lupus erythematosus (SLE) continue to represent a serious challenge for health care professionals. This fact is particularly true of SLE, a sys- temic autoimmune disease that affects multiple organ systems in- cluding the skin (e.g., dermatitis), joints (e.g., rheumatoid arthritis), brain/CNS, blood vessels (e.g., vasculitis, atherosclerosis, arterioscle- rosis), cardiac tissues and most severe and often fatal, the kidneys (e.g., glomerulonephritis (GN)), sclerosis, end-stage renal disease (ESRD). SLE mostly affects women of childbearing age and certain mi- norities disproportionately, with a prevalence of several hundred thousand patients with lupus in the US alone [1,2].
Treatment of SLE is a daunting task as few options exist for patients especially those that have developed later stage complications such as lupus nephritis (LN), a chronic inflammatory disease of the kidneys that is fatal if not resolved [3,4]. One of the hallmarks of SLE is the pres- ence of antinuclear antibodies (ANA), most important, anti-double stranded DNA (dsDNA) antibodies, to which the later has been tightly linked to the development of fatal LN [5–7]. Current therapies for SLE include aggressive doses of immunosuppressive drugs such as high-
Abbreviations: (SLE), systemic lupus erythematosus; (Mab), monoclonal antibody.
⁎ Corresponding author. Tel.: + 1 610 738 6752; fax: +1 610 738 6755.
E-mail address: [email protected] (M.M. Seavey).
dose glucocosteroids (Dexamethasone (DEX), Methylprednisolone) or antimalarials (Hydroxychloroquine) [4,8]. Cyclophosphamide (CTX), Azathioprine (AZA) and Mycophenolate mofetil (MMF) are leading therapies to treat developing lupus nephritis, however, all three drugs are toxic and are used as maintenance therapies during flares [8,9]. Since autoantibodies are thought to be the contributing root of this disease and inflammatory factors increase the chances of SLE (e.g., cytokines and pathogens) it is postulated that targeting the plasma cells (that produce these autoimmune antibodies) may be a plausible target for the treatment of SLE [10–12].
The recent failures of Rituximab (anti-human CD20) B-cell deple- tion studies for the treatment of lupus and LN may be explained by the fact that PCs lack the expression of CD20 and thus are not suscep- tible to deletion by Rituximab [13]. However, use of the anti-IL-6R an- tibody, Tocilizumab (Roche) or the now approved anti-BLyS/BAFF antibody, Belimumab (HGS/GSK), target essential cytokines/growth factor pathways required for PC differentiation and proliferation [14–17]. Plasma cells are also very sensitive to changes in protein load and ER stress as these cells are responsible for producing very large quantities of soluble immunoglobulin [18].
Blockade of the cellular proteasome can lead to apoptosis via the activation of the unfolded protein response (UPR) due to ER stress [19]. Blockade of the cellular proteasome can also inhibit the activa- tion of NF-κB via the cytoplasmic accumulation of its negative regula- tor, IκBα [20]. Both mechanisms are central to the pathogenesis of SLE. Plasma cells produce much of the autoantigen specific
1567-5769/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.intimp.2011.11.019
antinuclear antibodies (ANAs) that are responsible for several patho- logical manifestations associated with SLE (e.g., glomerulonephritis, cardiomyopathies, dermatitis) [21]. Activation of the NF-κB cascade leads to several proinflammatory cytokine cascades that perpetuates the proliferation and differentiation of autoantigen specific T and B cells and drives the initial innate immune response that potentiates lupus-like symptoms in the early in disease [22].
The best known proteasome inhibitor is the peptidyl boronic acid, bortezomib or Velcade® (previously known as PS-341 or MLN-341). Bortezomib reversibly inhibits the chymotrypsin-like activity of the proteasome and is an approved proteasome inhibitor for the treat- ment of patients with multiple myeloma and mantle cell lymphoma and is currently in a Phase IV for the treatment of lupus nephritis pa- tients (www.clinicaltrials.gov) [23]. In preclinical mouse models bor- tezomib has shown efficacy in the treatment of RA [24] and SLE [12]. However, bortezomib has serious side effects in multiple myeloma (MM) patients [25], including polyneuropathy (36%), thrombocyto- penia (35%) and gastrointestinal complaints (57%) [26]. Similarly, PR-957, another proteasome inhibitor has demonstrated efficacy in both RA [27] and SLE mouse models [11,28]. Based upon these proof-of-concept studies, investigations with the proteasome inhibi- tor delanzomib were initiated in two mouse models of human SLE.
Delanzomib (CEP-18770) is a novel reversible P2 threonine bo- ronic acid proteasome inhibitor [20]. Delanzomib can promote apoptosis in human multiple myeloma (MM) cell lines and patient derived cells; inhibit endothelial cell survival, vasculogenesis and osteoclastogenesis in vitro and displays favorable cytotoxicity profile toward normal cells [20]. Both intravenous and oral administration of delanzomib are well tolerated and result in sustained pharmacody- namic (PD) and antitumor activity and survival benefits in xenograft and systemic models of human MM [20]. In this report we describe the use of the proteasome inhibitor delanzomib to treat lupus nephri- tis in two mouse models of SLE (i.e., MRL/lpr and NZB/WF1 (NZM) hy- brid) compared to standard of care and the reference proteasome inhibitor, bortezomib. Treatment with delanzomib decreased serum levels of several lupus-associated cytokines and antinuclear anti- bodies, especially that of the nephritogenic anti-dsDNA antibodies. The severity of SLE disease manifestations was ameliorated by treat- ment with delanzomib, particularly the development of immune- complex mediated glomerulonephritis (GN) and fatal renal disease. Delanzomib extended survival greater, relative to that of bortezomib and cyclophosphamide treatment and improved several immunolog- ical parameters including a reduction of pathogenic spleen PCs and a reduction in the severity of SLE histopathology. These data provide a compelling rationale for the clinical evaluation of delanzomib for the treatment of lupus patients, especially for lupus nephritis pa- tients’ refractory to standards of care or with relapsing GN.
2. Materials and methods
2.1. Animals and facilities
The in-life portions of these experiments were performed at Cephalon, Inc. (West Chester, PA), an AAALAC accredited facility. Six week old lupus-prone MRL/lpr (Jackson Labs, #000485) and non- lupus prone control MRL/MpJ (Jackson Labs, #000486) female mice were maintained on a 24 hour light/dark cycle, with food and water available ad libitum. NZBWF1/J (catalog no. 100008) and NZW/LacJ (catalog no. 001058) female SLE-prone, lupus nephritis positive (30–100 mg/dL) mice were obtained from Jackson Laboratories (Bar Harbor, ME) at 6-weeks of age. Animals were maintained on a 24 hour light/dark cycle, with food and water available ad libitum. Ex- perimental procedures were approved by and in accordance to the regulations of the Institutional Animal Care and Use Committee (IACUC) of Cephalon, Inc.
2.2. Compounds
Delanzomib (CEP-18770) (96.1% pure anhydrous/solvent free) and bortezomib (99.8% purity) were synthesized at Cephalon, Inc. Dexamethasone (DEX) (10 mg/mL) and cyclophosphamide (CTX) was purchased from Hanna’s Pharmaceuticals (Wilmington, DE). Ve- hicle used for the suspension of delanzomib and bortezomib was 87% PBS, 3% DMSO, 10% Solutol (Mutchler Inc., Solutol HS 15). Delanzomib and bortezomib were stored in 75 μL of DMSO at −80 °C in individual
single use aliquots.
2.3. Study design
MRL/lpr model: see Fig. S1A for complete experimental design lay- out and group definitions. MRL/lpr mice were randomized and initial bleeds collected, and body weights recorded for baseline measure- ments. All mice were individually ear tagged and monitored through- out the entire experiment. Mice were age-matched and treatment started at 6–8 weeks of age. NZM model: see Fig. S1B for complete ex- perimental design layout and group definitions. Age-matched, female, NZM or NZW/LacJ, mice were matured for the study of lupus nephritis for a total 7 months or 210 days at which time urine was collected for detection of proteinuria. Mice with 0.5–1.0 mg/mL of urine protein as determined by an optical density-based total protein precipitation assay or 30–300 mg/dL of protein as determined by a stick assay were considered proteinuria positive and selected for entry in the study. Groups were normalized to contain a Gaussian distribution of proteinuria positive animals (i.e., 1/3 low proteinuria, 1/3 medium, 1/3 high) and randomized between groups before ear marking and taking baseline measurements including urine and serum collections. A total of five mice from the total population were randomly selected for baseline kidney histology evaluation. Treatments and tests for each group are as described in Fig. S1B. Treatments were initiated at 212 days of age, some mice died shortly after treatment but all ani- mals regardless of health status were counted against the total group size from time of dosing. Of note, all graphs shown in these studies indicate day-0 as being the “start of treatment” and thus rep- resent 212 days of age. Day-98 or “end of treatment/study” repre- sents 310 days of age for the NZM animals. All graphs show time as “day on study” with day on study starting at 212 days of age and end- ing at 310 days of age. Mice were first randomized, initial bleeds col- lected, and body weights recorded for baseline measurements [see Fig. S1B].
2.4. Antinuclear antibody (ANA) ELISA assays
The measurement of serum anti-dsDNA and anti-Smith antibody (Ab) was performed by an in-house developed custom ELISA assay described elsewhere [29]. Chromatin-coated plates were purchased from Inova Diagnostics, Inc. Purified bovine thymus dsDNA (Sigma, St. Louis, MI) or purified bovine Smith antigen (GenWay, San Diego, CA) were used as coating antigen for the detection of anti-dsDNA and anti-Smith Ag Ab respectively. Coated plates were washed with Borate Sulfate Saline (BSS) and blocked with BSS containing 1% Bo- vine Serum Albumin (BSA) and 0.1% Tween-20 detergent. Standard curves were generated using mouse anti-chromatin Ab (Sigma, 2B1) or 25 week old MRL/lpr serum. Mouse anti-dsDNA Ab (Abcam, Cam- bridge, MA), or mouse anti-Smith antigen Ab (Abcam) was used as standard for each assay. Secondary Ab was purchased from Abcam (goat anti-mouse pAb-HRP), the substrate was purchased from Rock- land (Gilbertsville, PA) tetramethylbenzidine (TMB), and stop reac- tion buffer was generated by diluting 1 mL of concentrated sulfuric acid into 20 mL of dH2O. Developed plates were read using a Victor- X4 spectrophotometer reading at 450 nm with a reference wave- length of 570 nm.
2.5. Antibody secreting B-cell Elispot assays
Complete media (R10) was used for all experiments involving the ex vivo culture of splenocytes for Elispot experiments. Complete media consisted of RPMI1640 (Cellgro, Manassas, VA), 1% Pen-Strep (Cellgro, Manassas, VA), 1% L-Gln (Cellgro, Manassas, VA), 1% non-essential amino acids (NEAA) (Cellgro, Manassas, VA), beta-mercaptoethanol (β-ME) (Cellgro, Manassas, VA), and 10% fetal bovine serum (FBS, Cellgro, Manassas, VA). B-cell Elispot components were ordered from MabTech (Nacka Strand, Sweden) and nitrocellulose IP filter plates obtained from Millipore (Billerica, MA). Elispot wells were coat- ed with either purified bovine thymus dsDNA (Sigma), purified bovine Smith Ag (GenWay) or boiled filtered purified chicken chromatin from lysed chicken red blood cells (Rockland, Gilbertsville, PA) at 10 μg/mL. Spleens were processed using glass homogenization, fil- tered through a 60 μm sterile cell strainer and red blood cells (RBCs) lysed using BioLegend lysis buffer (San Diego, CA). Processed spleno- cytes were added to each well in culture medium. Anti-mouse pan- IgG was used as a positive control for total IgG producing antibody se- creting cells (ASC) and used to normalize results.
2.6. Luminex analysis of serum cytokine samples
Cytokines were measured using the mouse cytokine 10-plex (MRL/lpr) or 20-plex (NZM) bead kit (Invitrogen, Carlsbad, CA, no. LMC0001 (10-plex), LMC0006 (20-plex)). Filter plates (Millipore, Billerica, MA, no. MAIPSWU10), were pre-wet with 200 μL of wash so- lution (kit component) and 25 μL of beads were added per well. Serum samples were diluted and a total volume of 50 μL was added per well (i.e., 25 μL of sample serum plus 25 μL of assay diluent). Plates with beads were incubated for 2 h at room temperature (RT) on an orbital shaker in the dark. At the end of the incubation, plate
(s) were washed twice in buffer, secondary biotinylated Ab was added at a 1:10 dilution (100 μL) in biotin diluent provided with the kit. Plates were incubated at RT for 1 h in the dark then washed twice in buffer. Streptavidin in assay diluent was added at 100 μL per well, then incubated for 30 min at RT in the dark. The plates were washed 3 times then 100 μL of wash solution was added and ag- itated for 2–3 min at RT in the dark. Plates were run immediately fol- lowing this incubation period on a Luminex xMAP 200 unit with data acquisition and analysis software (Invitrogen, San Diego, CA, no. MAP0200). All bead washing was performed using a vacuum mani- fold unit (Pall, Ann Arbor, MI no. 5017). For all cytokine Luminex as- says, values below the limit of detection set by the lowest point along the standard or set by the manufacturer were considered out of range and were not estimated, but assumed the lowest point along the stan- dard curve for that particular assay.
2.7. Urinalysis
Urine samples were acid precipitated for total protein recovery and analysis using the rodent urinalysis kit (Chondrex, no. 9040). Samples for which sufficient urine remained were used for Uristix® leukouria analysis for confirmation of proteinura. A standard protein solution was prepared from normal mouse sera and was used as a standard for mouse urinary protein assay by turbidity. Standard prep- aration was as follows: 0, 5, 10, 15, 20, 30, 40, and 50 μL of a 4 mg/mL mouse sera standard protein solution were added in duplicate into two columns. PBS was added to individual wells to adjust the final volume to 50 μL. For urine sample preparation, urine samples were centrifuged at 9880 ×g for 3 min using a tabletop micro-centrifuge. Urine supernatant (1–50 μL) was added in duplicate. PBS was added to adjust the volume to 50 μL total. For the turbidity assay: 25 μL of
0.1 N HCl was added into blank columns and 250 μL of 3% sulfosali-
cyclic acid into the test columns. The microplate was incubated for 10 min at RT and plates were read using an ELISA reader with single
beam at 450 nm. For the Uristix® strip assay 20 μL of urine was placed onto each test strip square and incubated for at least 30 s before the result was recorded. For all total urine analysis plate-based assays, values below the limit of detection set by the lowest point along the standard or set by the manufacturer were considered out of range and were not estimated, but assumed the lowest point along the stan- dard curve for that particular assay.
2.8. Histology
For histological analyses, the left kidney from each animal was re- moved and fixed in 10% buffered formalin for 48 h on an orbital rock- er at 25 °C, then washed overnight with running dH2O and stored in 70% ethanol at 4 °C until ready to be processed. Kidney stains used for histological analyses included Hematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS) and Trichrome stains (Wistar Institute, Philadelphia, PA). Only H&E stained sections are shown in this report; however, all 3 stained sections were used by the pathologist for scor- ing purposes. All histological scorings were performed blinded and by an independent board certified veterinary medical pathologist (GetA- Path Consulting LLC, New Bolton, PA). Multiple stains were used to determine score values. Images show the worst affected area of sec- tion selected blindly by the pathologist. Kidney IC-GN Scoring Method [30]: glomerular cellularity, glomerular necrosis, glomerulosclerosis, interstitial infiltration, tubular atrophy, interstitial fibrosis, vasculitis were all given a score of 1–5 for null, low, moderate, high, or severe disease, respectively (see Supplemental Material and methods sec- tion for detailed definitions of score parameters).
2.9. Pharmacodynamic (PD) analysis of proteasome activity
Spleen and kidney were collected on day 118 (MRL/lpr model) or day 98 (NZM model) 3 h post final dose administration and snap fro- zen on dry ice with cooled isopentene and stored at −80 °C. Lysed spleen and kidney were processed using the following protocol.
Seven hundred microliters of tissue extraction buffer containing pro- tease inhibitor cocktail (Calbiochem, no. 539136) and Halt phospha- tase inhibitor cocktail (Thermo scientific, no. 78420) or Roche phosphatase inhibitor cocktail (Roche, no. 04906837001) in tissue ex- traction reagent I (Invitrogen, no. FNN0071) was added to each sam- ple. Samples were homogenized frozen using a PT 10–35 Polytron homogenizer (VWR, no. 97036-082). After homogenization the sample was centrifuged at 4 °C for 2000 ×g for 10 min, supernatant was re-centrifuged at 4 °C at maximum speed, 14,000 ×g, for 15 min. Supernatants were carefully removed to avoid picking up the top layer of lipids/adipose debris. Protein concentration was adjusted to 3 mg/mL using a BCA protein assay (Pierce, no. 83228). Spleen protea- some activity was analyzed using 20S fluorogenic assay (Cayman Chemical Company, Cat#10008041, Loat#0414698-1) and modula- tion of proteasome activity in the kidney was analyzed using the I Bα accumulation ELISA assay (Cell Signaling, Cat#7355, Lot#17). Both assays were performed per manufacturer’s instructions.
2.10. Data and statistical analyses
Significance for parameters measured over time was determined primarily by 1-way ANOVA tests. For individual comparisons, area under the curve (AUC) values were determined for each group with parameters measured over time and AUC values for individual groups as compared to the vehicle were used to determine magnitudes over or under the vehicle AUC. This allowed the analysis of all fluctuations in data over time when comparing statistical significance using the 1- way ANOVA test for the same data set. All ELISA and Luminex® assays were evaluated using linear regression analyses to determine concen- tration of analyte following data acquisition. Mann–Whitney non- parametric and 1- or 2-way ANOVA were used as statistical tests
where noted in figure legends depending on the experiment and test- ed hypothesis. A p-value less than 0.05 was considered significant. Statistical software used was Graph Pad Prism (vs. 5.01, 2007), calcu- lations were performed using Microsoft Office Excel (Professional, 2003).
3. Results
3.1. Treatment of MRL/lpr lupus mice with delanzomib reduces the level of circulating proinflammatory cytokines and disease-promoting antinuclear antibodies
Serum cytokines were measured as an indicator of disease treat- ment response. Numerous cytokines are elevated in lupus patients, the most common is type I interferon (IFNαβ) [31]. In addition, IL- 12 and IL-10 are also elevated and can be used as prognostic markers of disease progression [32,33]. SLE is regarded as a systemic autoim- mune disease and thus several of the common pro-inflammatory cy- tokines are also elevated during the course of disease such as IL-1β and TNFα, [34], both of which were monitored during MRL/lpr mouse disease progression (Fig. 1).
Both delanzomib and bortezomib treatment reduced serum IL-12 levels in the MRL/lpr model below that of vehicle-treated animals (48%, 72%, and 62% decrease in serum IL-12 levels below that of vehicle-treatment for groups 1–3 respectively, and a decrease of 54% by groups 4–5 compared to vehicle-treatment at end-of-study (EOS) day 118) (Fig. 1A). Serum IL-1β was reduced by delanzomib 3 mg/kg iv twice weekly administration as compared to vehicle trea- ted animals (16.8 pg/mL down to 5.7 pg/mL by EOS) (pb 0.05, Fig. 1B). Serum TNFα was reduced by both delanzomib and bortezo- mib treatment as compared to vehicle-treated animals (62%, 79%, 71%, 64%, and 71% decreases in serum TNFα as compared to vehicle by end of study for groups 1–5 respectively) (Fig. 1C).
The presence of anti-dsDNA Ab is used as a clinical biomarker as-
sociated with a poor prognosis of lupus and is strongly associated with the development of nephritis [35,36]. Hence, serum samples of treated MRL/lpr mice were analyzed for the presence of circulating pathogenic antinuclear antibodies (ANA). Anti-dsDNA IgG ANA levels were significantly reduced below vehicle-treated animals only for the two delanzomib (iv) treatment groups (80% and 84% decreases for groups 1–2, respectively) and for both bortezomib treatment groups (82% and 58% decreases for groups 4–5, respectively) (Fig. 2A). Simi- lar responses were observed for anti-Smith Ag IgG ANA levels (97%, 100%, 86%, and 74% decrease for groups 1–4 compared to vehicle- treated animal at EOS) (Fig. 2B). Delanzomib treatment reduced the levels of anti-chromatin IgG below that of the vehicle-treated animals (Fig. 2C; pb 0.001) for all 3 treatment groups (89%, 98%, and 79% for groups 1–3 respectively); only bortezomib 0.5 mg/kg iv once weekly treatment reduced levels below vehicle-treated animals (64% de- crease) (Fig. 2C; pb 0.001).
Antinuclear antibodies are produced by both proliferating B cells but also that of short and long-lived plasma cells (LL-PCs) [21]. The later produce most of the circulating ANAs and primarily resides in the bone marrow, sites of inflammation and spleen [37]. Consequent- ly, therapy that impacts the longevity of long-lived plasma cells will ultimately impact the circulating levels of pathogenic antibodies and thus disease. Circulating autoantibody secreting cell types (ASCs) are directly correlative to a poor SLE prognosis [10,12,38]. With this prior knowledge, ASCs were measured in the spleens of treated MRL/lpr mice using the sensitive cell-based ex vivo Elispot assay. All
3 delanzomib treatment regimens reduced the frequency of anti- chromatin IgG-producing spleen ASCs below that of both dexameth- asone and vehicle groups (Fig. 3A); both delanzomib iv treatment groups reduced frequencies below that of bortezomib 0.3 mg/kg 2 × a week iv (Fig. 3A; pb 0.05). Treatment with delanzomib reduced the frequency of anti-Smith Ag and anti-dsDNA IgG producing ASCs
for all three delanzomib treatment groups as compared to bortezomib
0.3 mg/kg iv twice weekly treated animals (97%, 100% and 86% de- crease for Smith Ag; 80%, 84%, 82% decrease for dsDNA ASCs as com- pared to vehicle treatment for groups 1–3) (Fig. 3B).
Due to the hyperproliferation of T and B cells in SLE, both spleno- megaly (spleen swelling) and lymphomegaly (lymph node swelling) are common in this model (MRL/lpr). Control of systemic autoimmune responses may involve reduction in general lymphocyte numbers and thus spleen and lymph node size is important to note during any treat- ment. The reduction in percentage of lymphomegaly-positive mice was significant for both the delanzomib 3 mg/kg iv once weekly and twice weekly treatment groups over that of the vehicle treatment group (71% and 34%, pb 0.05 decreases, respectively) (Fig. S2A). Spleen weights of 25 week old mice were determined as a measure of spleno- megaly. A reduction of spleen mass was observed for all treatment groups as compared to the vehicle treatment group (568 mg down to 150–300 mg by end of study) (pb 0.01) (Fig. S2B). The greatest decrease was observed for the delanzomib 3 mg/kg iv twice weekly treatment groups as compared to the vehicle treatment groups at the end of the study (74% decrease, pb 0.001) (Fig. S2B).
3.2. Delanzomib protects MRL/lpr lupus mice from developing fatal lupus nephritis
The presence of serum anti-dsDNA Ab along with the presence of autoantibody secreting cell types are both correlated with an in- creased likelihood of developing lupus nephritis [12,35]. Lupus ne- phritis consists of several overlapping renal pathologies that most commonly include glomerulonephritis, necrosis and sclerosis associ- ated with chronic renal inflammation. Renal damage is monitored in the clinic by changes in serum creatinine, increases in urine protein (proteinuria) and evaluation of blood or leukocytes in the urine (hemauria and leukouria, respectively). In treated MRL/lpr mice all three delanzomib treatment regimens significantly reduced urinary protein levels below that of vehicle controls (60%, 70%, and 71% for groups 1–3 respectively) (pb 0.01), whereas only 0.5 mg/kg iv borte- zomib twice weekly reduced proteinuria significantly below vehicle (55% decrease) (pb 0.05) (Fig. 4A). Both delanzomib and bortezomib treatment also reduced leukouria levels below that of vehicle con- trols (69–96% decrease compared to vehicle treatment) (pb 0.001) (Fig. 4B).
Proteinuria is the direct result of renal damage and the later eval- uated by histopathology and scored by a board certified pathologist using the scoring method described by Smith et al. and outlined in the Methods and Material section [7]. The remaining mice at the end of this 25 week experiment were analyzed for the presence and extent of glomerulonephritis by conventional histology methods. All three delanzomib treatment regimens reduced the severity of the renal histopathology profiles as compared to vehicle-treated mice (1.5–1.8-fold decrease in average score as compared to vehicle) (Fig. 4C and Table 1). Only bortezomib treatment at 0.5 mg/kg twice weekly reduced renal histopathology scores below that of vehicle treated controls (1.6-fold decrease in average score as compared to vehicle) (Fig. 4C and Table 1). Most importantly, all three delanzomib treatments reduced renal interstitial infiltration up to 61% over that of the vehicle treatment as compared to a maximum of 47% by bortezo- mib (Fig. 4C and Table 1).
Both the 20S proteasome assay and an IκBα accumulation ELISA were used to measure the pharmacodynamic (PD) activity of delan- zomib in the spleen and kidneys of treated mice. All three delanzomib treatment regimens decreased significantly 20S proteasome activity compared to vehicle controls in the spleen of treated mice (40%, 45% and 41% decreases for groups 1–3, respectively) (pb 0.01) (Fig. S2C). Similar findings were observed for bortezomib treatment (40% and 41% decreases relative to vehicle treatment, respectively for groups 4 and 5). In the kidney, where active and often fatal disease
A Serum IL-12
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Fig. 1. Serum cytokines from MRL/lpr lupus mice treated with delanzomib (CEP-18770). MRL/lpr mice were treated as outlined in the legend. Graph shows Mean ±SEM for the concentration of mouse serum IL-12 (A), IL-1β (B) and TNFα (C) from treated MRL/lpr mice. Cytokines were analyzed using Luminex bead kits. Statistics used for comparisons was 1-way ANOVA.
is precipitated, proteasome inhibition was of greater magnitude as measured by an IκBα accumulation assay, as both delanzomib and bortezomib treatment led to an accumulation of cytoplasmic IκBα 3–6 fold over that of the vehicle treatment group (266–537% in- creases over vehicle) (pb 0.001) (Fig. S2D).
3.3. Delanzomib can reverse lupus nephritis in NZM mice
Both once and twice weekly administration of delanzomib re- duced proteinuria of treated mice to a greater extent than that of once weekly bortezomib, dexamethasone, and vehicle (61% and 72%
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Fig. 2. Serum antinuclear antibody titers from MRL/lpr lupus mice treated with delanzomib (CEP-18770). Treated MRL/lpr mouse serum samples were analyzed for the presence of anti-dsDNA (A), anti-Smith (B) or anti-Chromatin (C) IgG circulating ANAs via ELISA assay (see Materials and methods). Graph shows Mean ±SEM of ANA concentration in ng/mL, 100-fold dilution (A and B), and 2000-gold dilution (C) from original stock, statistics performed was 1-way ANOVA.
for groups 1–2 as compared to vehicle) (pb 0.01) (Fig. S3A). End- stage lupus nephritis was evaluated by histopathology and scored by a board certified pathologist for the assessment of total renal dam- age in diseased animals. Both once and twice weekly administration of delanzomib decreased several renal pathologies associated with lupus nephritis over that of once weekly bortezomib treatment but not twice weekly bortezomib treatment (Fig. 5 and Table 2). In
general, delanzomib treatment positively impacted renal tissue dam- age and inflammation greater than that of bortezomib treatment when compared to both the vehicle and standards of care treatments (e.g., 45–55% decrease in score for delanzomib over several parame- ters vs. 6–14% decrease in score for bortezomib as compared to vehi- cle) (Fig. 5 and Table 2). Both renal and lung infiltrates were observed in the vehicle-treated mice (Fig. 5A–B).
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OVA
Coated Well (10 g/mL)
Fig. 3. Spleen antinuclear antibody secreting cells from MRL/lpr lupus mice treated with delanzomib (CEP-18770). (A) MRL/lpr treated mice spleens were processed for splenocytes for ex vivo Elispot assays. Elispot wells were coated with 10 μg/mL of smith antigen, dsDNA or ovalbumin protein. Fresh, whole splenocytes were added to each well at 500,000 cells per well in cell culture medium. (B) MRL/lpr treated mice spleens were processed for splenocytes for ex vivo Elispot assays. Elispot wells were coated with 10 μg/mL of boiled chick- en chromatin or ovalbumin protein. Fresh, whole splenocytes were added to each well at 50,000 cells per well in cell culture medium. (A–B) Cells were incubated overnight at 37 °C. Developed wells provided spots that were counted as frequency of ASCs per million splenocytes. Statistics for comparisons included a two-tailed Mann–Whitney t-test. Single out- lier value greater than 2.5 times the standard deviation in vehicle group, were excluded from the analysis for both dsDNA and Smith antigen. Graphs show Mean ±SEM.
3.4. Treatment of NZM lupus nephritis mice with delanzomib reduces the level of circulating proinflammatory cytokines, disease-promoting antinuclear antibodies and ASCs
Serum IL-12 was significantly decreased upon treatment when com- pared to the vehicle group for both once and twice weekly doses of delanzomib (83% and 67% decrease respectively) (pb 0.01) (Fig. S3A). For both delanzomib treatment groups serum IL-12 levels were de- creased to a greater extent than bortezomib as compared to the vehicle (67–82% decrease for delanzomib compared to 32–54% decrease for bortezomib) (Fig. S3A). Administration of delanzomib twice weekly de- creased serum anti-chromatin Ab (49% decrease compared to vehicle) (Fig. S3B). Once weekly administration of delanzomib decreased serum anti-chromatin Ab greater than that of DEX and vehicle (63% de- crease) (pb 0.05) (Fig. S3B). Once and twice weekly administration of delanzomib decreased anti-chromatin ASC frequencies below that of bortezomib once and twice weekly treatment (pb 0.01 for 1× a week delanzomib; pb 0.05 for 2× a week delanzomib) and that of DEX (pb 0.01) and vehicle (pb 0.01) (100–96% decrease in anti-chromatin ASC for delanzomib as compared to vehicle) (Fig. S3C). Similar results
were observed for total IgG producing ASC in the spleen of treated mice (data not shown).
3.5. Delanzomib treatment extends the survival of mice with lupus nephritis
Lupus-prone mice usually die from end-stage renal disease (ESRD) manifested as nephritis. Consequently both mouse survival and body weight are necessary to monitor the impact of treatment but also the extent to which disease has on the growth of the animal. Treat- ment of SLE-prone, proteinuria-positive NZM mice with delanzomib extended survival significantly over vehicle (55% and 46% respective- ly; pb 0.001) and significantly extended survival over that of both CTX and both bortezomib treatment groups (pb 0.01) (Fig. S3D) (pb 0.001). Both 20S proteasome activity and IκBα accumulation were used as pharmacodynamic indicators of delanzomib-mediated proteasome inhibition in the spleen and kidneys of treated NZM mice. Once and twice weekly administration of delanzomib inhibited spleen 20S proteasome activity as compared to vehicle treatment (pb 0.05) (40% inhibition relative to vehicle) (Fig. S4A). Twice,
A Urine Leukocytes
4
B Total Urine Protein
3
2
1
0
71 85 99 118
Day of Study (18-25wks age)
0 25 50 75 100 125
Days Post-Treatment (8-25wks)
C
Fig. 4. Proteinuria and renal histopathology of MRL/lpr lupus mice treated with delanzomib (CEP-18770). (A) Urine collected from treated MRL/lpr mice was analyzed for total protein content using a rat urinalysis kit. Graph shows Mean ± SEM of protein concentration in mg/mL, statistical test used was 1 way ANOVA. (B) Urine from treated MRL/lpr mice was tested using Uristix assays for the presence of leukocytes or leukoria. Graph shows score given to each strip according to the manufacturer’s instructions for the last 4 time points for the study. Graphed result is Mean ±SEM, statistical test used was 1 way ANOVA. (C) Renal histology from treated MRL/lpr mice: H&E stained paraffin wax embedded kidney tissue sections from end-of-study mice. Images show most severely affected area of tissue sections selected blindly by the pathologist. ♠a, Normal kidney, no inflammation/pathology; ♣a, Normal glomerulus and size, no pathology; ♦a, Vasculitis, ateriosclerosis; ♥a, Interstitial infiltration, glomerulonephritis; ★a, Glomerular hypercellularity, parietal cell hyperplasia/hypertrophy; ●a, Glomerular deposits, enlarged glomerulus, membranous nephropathy.
Table 1
Renal scores from delanzomib (CEP-18770) treated MRL/lpr mice. Pathologist scored H&E, PAS and Trichrome stained paraffin-wax embedded kidney sections displayed as mean± SD, N = 5 mice per group shown, statistics were performed using a two tailed Mann–Whitney t-test, *p b 0.05, **p b 0.01 and ***p b 0.001. Pathologist scoring method and definitions can be found in the method and materials section.
MRL/lpr Renal Scores (Mean±SEM) Glomerular cellularity Glomerular necrosis Glomerulo sclerosis Interstitial infiltration Tubular atrophy Interstitial
fibrosis Vasculitis
CEP-18770 3 mg/kg 1 × a week iv 2.33 ± 0.51*** 2.00 ± 0.00** 2.00 ± 0.00** 2.16 ± 0.40*** 1.66 ± 0.51 1.83 ± 0.40 2.16 ± 0.40**
CEP-18770 3 mg/kg 2 × a week iv 2.54 ± 0.68*** 1.72 ± 0.46*** 1.81 ± 0.63*** 1.54 ± 0.52*** 1.36 ± 0.50*** 1.54 ± 0.52** 1.81 ± 0.60***
CEP-18770 10 mg/kg 2 × a week po 2.60 ± 0.20*** 2.20 ± 0.20** 2.20 ± 0.20** 1.90 ± 0.20*** 1.50 ± 0.30** 1.90 ± 0.20 2.30 ± 0.10**
Bortezomib 0.5 mg/kg 1 × a week iv 2.71 ± 0.48** 2.28 ± 0.48* 1.85 ± 0.69*** 2.00 ± 0.57*** 1.28 ± 0.48** 1.49 ± 0.53** 1.85 ± 0.69***
Bortezomib 0.3 mg/kg 2 × a week iv 3.28 ± 0.75 2.42 ± 0.78 2.71 ± 0.48 2.28 ± 0.75*** 1.85 ± 0.69 1.85 ± 0.37 2.57 ± 0.53
Vehicle 2 × a week po 3.88 ± 0.92 3.22 ± 0.97 3.33 ± 1.32 3.77 ± 0.97 2.55 ± 0.72 2.55 ± 0.88 3.33 ± 1.00
Compared to vehicle *p b 0.05** p b 0.01 ***p b 0.001 2-way ANOVA
but not once, weekly administration of delanzomib increased the ac- cumulation of kidney I Bα levels above that of the vehicle-treatment (40% increase, pb 0.05) (Fig. S4B).
3.6. Delanzomib can be provided both i.p. and s.c. and treats nephritic mice in a dose-dependent manner
In additional studies, different routes of administration were test- ed to determine the lower limit of activity for delanzomib in treating lupus nephritic mice. Additional studies were performed using 3, 1 and 0.3 mg/kg of delanzomib provided both a systemic (i.p.) route and local (s.c.) route. This was matched with 0.3 mg/kg twice a week dose of bortezomib provided i.p. or s.c. Standard of care agent included CTX and non-lupus control mice included the parent NZW/LacJ strain. Subcutaneous administration of delanzomib was inferior to systemic
i.p. administration at the lower dose of 0.3 mg/kg but was equivalent at 3 mg/kg to extend the survival of treated nephritic NZM mice (Fig. 6A; *pb 0.05). Survival was on par with that of bortezomib and the highest dose of delanzomib used i.p. (3 mg/kg) provided 100% sur- vival benefit, equivalent to CTX (Fig. 6A; *pb 0.05). Prolonged survival of delanzomib treated NZM mice correlated with reduced proteinuria levels of treated mice (Fig. 6B; *pb 0.05) and improved renal histopa- thology (data not shown). Similar dose-dependent reductions in anti-
Smith (Fig. 6C; *pb 0.05) and anti-dsDNA ANAs (Fig. 6D; *pb 0.05) fol- lowed treatment and corresponding spleen anti-Smith (Fig. 7A;
*pb 0.05) and anti-dsDNA ASCs (Fig. 7B; *pb 0.05). Both ANAs and ASC levels fell to non-lupus (NZW/LacJ; bottom dotted line) levels and were well below the vehicle controls (upper dotted line for ASC
graphs). Reduction of CD19 − B220 − CD138 + CD38 + spleen plasma cells correlated with observed health benefits of delanzomib in both a
dose and route of administration dependent manner (Fig. 7C). Percent of spleen plasma cells were reduced at highest dose of 3 mg/kg to non- lupus, NZW/LacJ, levels (Fig. 7C; lower dotted line) and were on par with CTX.
4. Discussion
The studies described in this report evaluated the efficacy of pro- teasome inhibition in pre-clinical models of early stage and progres- sive SLE. Treatment of both lupus-prone MRL/lpr and NZM mice with the reversible P2 threonine boronic acid proteasome inhibitor, delanzomib resulted in a significant improvement in survival, tolera- bility and reduction of several lupus-associated immune-parameters compared to both standards of care agents, dexamethasone, and cy- clophosphamide, and the proteasome inhibitor, bortezomib.
Fig. 5. Renal and pulmonary histopathology from NZM mice treated with delanzomib (CEP-18770). (A) Renal histology from treated NZM mice: H&E stained paraffin wax embed- ded kidney tissues sections from end-of-study mice. Images show worst affected area of section selected blindly by the pathologist. ♠a, Normal kidney, no inflammation/pathology;
♣a, Normal glomerulus and size, no pathology; ♦a, Vasculitis, ateriosclerosis; ♥a, Interstitial infiltration, glomerulonephritis; ★a, Glomerular hypercellularity, parietal cell hyper-
plasia/hypertrophy; ●a, Glomerular deposits, enlarged glomerulus, membranous nephropathy. (B) Representative images from H&E stained paraffin-wax embedded lungs from treated NZM mice. ♠a, Normal lung, no inflammation/pathology; Δa Adenoma; ♦a, Vasculitis, ateriosclerosis or pulmonary edema pathology. Lung samples were not scored, pa- thologist comments not shown.
Table 2
Comparisons table of renal scores, anti-dsDNA antibody titers and proteinuria levels for delanzomib (CEP-18770) Treated NZM Mice. Table displays scores for individual mice for kidney sections stained with H&E, PAS and Trichrome stains. Individual mice are displayed for age (full life or EOS date), anti-dsDNA IgG titers in ng/mL concentrations, each scoring parameter with score represented by +/− marks with (0)/−=no disease, (1)/+=mild disease, (2)/++=moderate disease, (3)/+++=high disease, and (4)/++++=severe disease. Please see Materials and methods section for full description of each score and pathologist definitions. Proteinuria also shown in mg/mL concentrations from total urine
protein assays. Null values such as “0.00” were considered “below quantifiable levels” (BQL) for the assay. Statistics were performed using a two tailed Mann–Whitney t-test,
*p b 0.05, **p b 0.01 and ***pb 0.001.
Group Mouse Age (d) Anti-dsDNA IgG (ng/mL) Glomerular cellularity Glomerular necrosis Glomerular sclerosis Interstitial infiltration Tubular atrophy Interstitial
fibrosis Vasculitis x> 1 mg/mL bold proteinuria
(mg/mL)
Parental control NZW/LacJ-1 310 54.1 − − − − − − − 1.51
NZW/LacJ-2 310 125.0 − − − − − − − 1.61
NZW/LacJ-3 310 27.8 − − − − − − − 1.76
NZW/LacJ-4 310 65.9 − − − − − − − 1.00
Mean ±SD 310 ± 0.0 68.2 ± 41.0 1.47 ± 0.32
Vehicle 9A1 212 ND ND ND ND ND ND ND ND 3.58
9A2 240 51.4 ++ + − − − − + 8.64
9A3 310 32.0 ++ + +++ +++ +++ +++ + 1.64
9A4 268 ND +++ + + − ++ + − 13.44
9A5 310 16.7 + − − − − − + 0.85
9B1 310 95.2 ND ND ND ND ND ND ND 2.04
9B2 240 27.0 ++ + ++ + ++ ++ ++ 8.63
9B3 310 253.2 + + − + + − + 1.17
9B4 227 ND +++ ++ +++ +++ +++ +++ ++ 12.67
9B5 227 57.0 +++ ++ ++ ++ + + + 7.48
9 C1 310 214.8 +++ + +++ +++ +++ +++ ++ 1.30
Mean ±SD 269.5 ± 41.0 93.4 ± 90.5 5.58 ± 4.7
CEP-18770 3mpk 1A1 310 5.9 + − − − − − − 0.82
1 × a week 1A2 310 20.2 − − − − − − − 1.26
1A3 310 8.8 − − − − − − − 1.35
1A4 310 46.9 − − − − − − − 1.87
1A5 310 21.6 + + − + − − − 0.88
1B1 310 23.4 + − − − − − − 1.47
1B2 310 122.0 + + − − − − − 1.39
1B3 310 23.0 + + − + − − − 0.81
1B4 310 22.5 − − − − − − − 0.82
1B5 310 32.5 ++ ++ ++ ++ + + − 1.97
1 C1 310 11.3 + + − − − − − 0.66
1 C2 310 12.7 − − − − − − − 0.53
Mean ±SD 310.0 ± 0.0 29.2 ± 31.2* 1.15 ± 0.47**
CEP-18770 3mpk 2A1 310 12.2 − − − − − − − 0.57
2 × a week 2A2 310 27.6 ND ND ND ND ND ND ND 0.83
2A3 310 10.4 − − − − − − − 1.76
2A4 310 27.9 + + + + + + + 1.31
2A5 268 ND − − − − − − − 0.95
2B1 310 51.8 ND ND ND ND ND ND ND 1.12
2B2 310 26.5 + + + + − − − 1.21
2B3 310 19.3 + + − − − − − 1.04
2B4 310 34.7 − − − − − − − 2.21
2B5 212 ND + − − + − − − 10.63
2 C1 310 53.1 + + − − − − − 3.13
2 C2 310 34.3 + + − + + − − 2.23
Mean ±SD 298.3 ± 29.7 29.7 ± 14.4 2.24 ± 2.7
Bortezomib 0.3mpk 5A1 310 71.3 ++ + + − + + + 5.16
1 × a week 5A2 254 26.8 ++ +++ +++ +++ ++ ++ + 7.22
5A3 227 15.2 +++ ++ +++ +++ +++ ++ + 10.68
5A4 310 18.2 ++ + + + − + − 1.31
5A5 310 20.2 ++ +++ +++ ++ +++ ++ + 13.31
5B1 254 ND + + +++ + +++ ++ + 10.96
5B2 310 390.0 + + + − ++ + − 3.95
5B3 268 ND ND ND ND ND ND ND ND 13.69
5B4 310 184.3 + − − − − − − 1.32
5B5 310 107.3 ++ + ++ − ++ ++ + 4.36
5 C1 291 16.3 +++ +++ +++ ++ ++ ++ ++ 9.87
5 C2 310 58.3 ++ + − + − − + 4.63
Mean ±SD 288.7 ± 29.8 90.79 ± 118.1 7.2 ± 4.3
Bortezomib 0.3mpk 6A1 212 61.1 ND ND ND ND ND ND ND 10.49
2 × a week 6A2 310 353.6 ++ + + − + + + 1.49
6A3 310 53.0 ++ + − − − − − 0.73
6A4 310 26.6 − − − − − − − 1.49
6A5 310 31.0 + + + − − + − 0.74
6B1 310 29.4 + − − − − + + 1.09
6B2 310 5.9 + + − − − − + 0.67
6B3 310 87.8 ++ +++ +++ ++ +++ +++ ++ 1.13
6B4 240 27.9 + + + − + + + 7.86
6B5 310 26.4 + + − − + − + 0.56
6 C1 310 164.6 + + + − − − − +2.97
6 C2 240 26.2 ++++ +++ +++ ++ +++ +++ ++ 10.12
Table 2 (continued)
Group Mouse Age (d) Anti-dsDNA IgG (ng/mL) Glomerular cellularity Glomerular necrosis Glomerular sclerosis Interstitial infiltration Tubular atrophy Interstitial
fibrosis Vasculitis x> 1 mg/mL bold proteinuria
(mg/mL)
Mean ±SD 290.2 ± 36.5 74.4 ± 97.5 3.27 ± 3.8
CTX 50mpk 7A1 227 13.4 ++ ++ +++ + ++++ +++ + 11.09
1 × a week 7A2 310 6.9 + − − − − − − 0.86
7A3 310 22.2 + + ++ − + − − 1.44
7A4 310 13.2 ++ + ++ +++ ++ + − 0.76
7A5 310 40.3 + − ++ − − + − 1.30
7B1 310 9.6 + ++ + − − − + 0.71
7B2 212 65.1 ND ND ND ND ND ND ND 8.84
7B3 310 52.4 + + − − + − − 0.96
7B4 240 24.3 − − − + + − − 0.58
Mean ±SD 7B5 310
284.9 ± 40.9 61.9
30.9 ± 22.2 − − − − − − + 1.50
2.8 ± 3.8
Dex 1.5mpk 8A1 291 27.2 ++ ++ +++ ++ +++ +++ + 8.43
3 × a week 8A2 212 18.9 10.19
8A3 291 20.0 ++ ++ +++ ++ +++ ++ + 11.02
8A4 240 79.5 ND ND ND ND ND ND ND 8.47
8A5 268 167.5 + + − − − − − 0.35
8B12 40 ND ND ND ND ND ND ND ND 10.19
8B2 212 27.8 ++ ++ +++ ++ +++ ++ + 7.16
8B3 310 16.2 + + ++ − − − + 0.55
8B4 310 10.9 +++ +++ ++ +++ +++ ++ +++ 12.36
8B5 240 17.1 +++ +++ ++++ +++ +++ +++ +++ 9.89
Mean ±SD 261.4 ± 37.6 42.7 ± 51.0 7.86 ± 4.1
However, for the NZM model, unexpected increases of several tested disease parameters above vehicle controls for the 0.3 mg/kg 1 × a week iv dose of bortezomib included splenomegaly, proteinuria, and ANA. A possible cause for this result is weak immunosuppression induced by the once weekly dose of bortezomib as these changes from vehicle for many assays were not significant. Increases in SLE mouse survival observed with delanzomib treatment paralleled the reductions in splenomegaly, ANA, ASCs and was consistent with the
reduced severity of multiple renal pathologies. Similarly bortezomib at the highest dose tested, 0.3 mg/kg iv twice weekly, provided dis- ease treatment with an overall reduction in several immune parame- ters. This was expected as both drugs share near identical mechanisms of action and can impact ongoing and most important, initiation, of new immune responses. For example, both delanzomib and bortezomib treatment increased the cellular accumulation of IκBα further decreasing nuclear pools of activated NFκB [20]. Weak
A B
12
CEP-18770 3 mg/kg 2x wk ip (100%)*
NZM Proteinuria
*p<0.05 vs. Vehicle, 1-w ANOVA
125
100
75
50
25
0
0 25 50 75 100
CEP-18770 1 mg/kg 2x wk ip (100%)* CEP-18770 0.3 mg/kg 2x wk ip (58%) CEP-18770 3 mg/kg 2x wk sc (100%)* CEP-18770 1 mg/kg 2x wk sc (81%) CEP-18770 0.3 mg/kg 2x wk sc (27%) Bortezomib 0.3 mg/kg 2x wk ip (88%) Bortezomib 0.3 mg/kg 2x wk sc (100%)* CTX 50 mg/kg 1x wk ip (100%)*
Vehicle 2x wk ip (25%)
10
8
6
4
2
*
0
0 28 58 72 91
Day of Study (0d = 7 mos old)
C Serum Anti-Smith ANA
Day of Study (0d = 7 mos old)
D Serum Anti-dsDNA ANAs
600
150
*p<0.05 vs. Vehicle, 1-w ANOVA
100
400
200
0
0 28 58 72 91
Day of Study (0d = 7 mos old)
50
*
0
0 28 58 72 91
Day of Study (0d = 7 mos old)
Fig. 6. Delanzomib (CEP-18770) treats lupus nephritis NZM mice via two routes of administration in a dose-dependent manner to improve survival and proteinuria. (A) NZM mice were treated as outlined in the legend. Graph shows percent of live mice for each week of the study. Statistics used for comparisons was a two-tailed paired student t-test. (B) Pro- teinuria levels of treated mice as determined by the rat urinary protein assay. Graph shows mean ±SEM; *p b 0.05, 1-way ANOVA. (C) Treated NZM mouse serum samples were analyzed for the presence of anti-Smith antigen and (D) anti-dsDNA IgG ANAs. (C–D) Graphs shows Mean ±SEM of ANA concentration in ng/mL, 100-fold dilution from original stock, statistics performed was 1-way ANOVA.
Spleen Anti-Smith ASCs
C
15
10
5
0
Groups
Fig. 7. Delanzomib (CEP-18770) treats lupus nephritis NZM mice via two routes of administration in a dose-dependent manner by reducing autoantibody producing plasma cells. (A–B) NZM treated mouse spleens were processed for splenocytes for ex vivo Elispot assays. Elispot wells were coated with 10 μg/mL of smith antigen (A), dsDNA (B). Fresh, whole splenocytes were added to each well at 500,000 cells per well in cell culture medium. Cells were incubated overnight at 37 °C. Developed wells provided spots that were counted as frequency of ASCs per million splenocytes. Statistics for comparisons included a two-tailed Mann–Whitney t-test. Graphs shows Mean ±SEM. (C) Flow cytometry analysis of whole splenocytes stained with CD19-FITC, CD45R/B220-Cyc, CD38-PE, CD138-APC and plotted as a percent of live size gated lymphocytes. Graphs show Mean±SEM; statistics for com- parisons included a two-tailed Mann–Whitney t-test.
to limited activation of this major T/B-cell activating transcription factor is necessary for IL-2 expression and the production of several pro-inflammatory and lupus-promoting cytokines [39]. Thus, the hyper-lymphoproliferation driven by the absence of Fas-mediated lymphocyte apoptosis combined with ongoing systemic autoimmune responses in the MRL/lpr model reduced upon proteasome-inhibitor treatment are most likely due to NF B inactivation via increased cyto- plasmic retention of accumulated intracellular I Bα.
Reduction of inflammatory cytokines driven by NF- B was expected upon proteasome inhibition and was observed for IL-12, IL-1β and TNFα as compared to vehicle for both delanzomib (all 3 groups) and bortezomib (0.3 mg/kg twice weekly dose only) treat- ment regimens [40]. However, changes in other cytokines relative to vehicle were not as pronounced including the IFN-inducible genes, CXCL9/MIG and CXCL10/IP-10, both of which were elevated in SLE pa- tients [41]. Similar results were observed for IL-13, IL-17A and TNFα [34,42]. Changes in serum cytokines appear to be mouse strain specif- ic, as reductions in TNFα were observed in the MRL/lpr model
(Fig. 1C). Cytokines contribute to the phenotypes observed in lupus, including the stimulation of B cells to mature into LL-PCs that contrib- ute to the majority of the ANAs found circulating in patients' serum. Due to the elevated production and secretion of immunoglobulin, plasma cells are very sensitive to ER stressors, notably, proteasome inhibition [12]. Blocking the cellular proteasome upregulates the UPR response and treatment of lupus prone mice with bortezomib in- duces the UPR response in plasma cells (i.e., the upregulation of sev- eral UPR proteins such as CHOP, XBP1, and GRP78/Bip) inducing apoptosis via a caspase-dependent mechanism [12]. Thus, it was hy- pothesized that both delanzomib and bortezomib treatment could impact the development of ANA-secreting plasma cells but also impact the levels of circulating ANA in the serum of treated animals. In the MRL/lpr model, both delanzomib and bortezomib treatments reduced levels of spleen ASCs (unknown if long- or short-lived plas- ma cells) and serum ANA, with all 3 regimens of delanzomib better in this regard to the highest dose of bortezomib (0.5 mg/kg iv once weekly) and comparable to the 0.3 mg/kg iv twice weekly dose of
bortezomib. This is more evident when comparing the increased ASCs found in the twice weekly 0.3 mg/kg bortezomib group and that of the ANA levels (Figs. 2–3). Greater ASC numbers in the spleen were correlated to increasing levels of ANA and depletion of spleen ASC by delanzomib led to reduced ANA levels overtime (Figs. 2–3). Similar to belimumab, delanzomib treatment reduced the number of circulat- ing ANA-producing plasma cells and reduce serum levels of circulat- ing ANA [12,17]. It is possible that both long-lived and short lived plasma cells contributed to the observed levels of serum ANA. The possibility that long-lived plasma cells are being observed in the cur- rent studies is greater due to the fact that all assays (including detec- tion of ASCs) used an anti-mouse IgG pan antibody, and it is thought that only long-lived and not short-lived plasma cells secrete IgG (i.e., secrete class-switched antibody) [43]. As a result of less circulating ANA, including anti-dsDNA, (the later which is associated with glo- merulonephritis and ESRD [5,44]), renal histopathology was reduced upon treatment with delanzomib and this agrees with the concomi- tant reductions in proteinuria and leukouria observed (Figs. 2 and 4). Reductions in serum ANA were most pronounced for the delanzo- mib treatment group as compared to vehicle treatment for anti-Smith and anti-chromatin Ab (IgG). However, despite the null change in anti-dsDNA Ab for any treatment group in the NZM model as com- pared to the vehicle, significant changes were observed in the renal biopsies and pulmonary manifestations ex vivo at the end of study (EOS) day 98 post-treatment (Fig. 5). From Elispot assays measuring total ASCs ex vivo, producing anti-chromatin Ab, when compared to total IgG producing ASCs, the decline in specific anti-pathogenic Ab was greatest for the delanzomib treatment groups suggesting selec- tivity or increased sensitivity of these expanded SLE-promoting plas- ma cells in NZM animals for delanzomib-induced cytotoxicity.
Bortezomib is not FDA approved to treat lupus or lupus nephritis and there are only a few manuscripts in the literature describing the use of bortezomib for autoimmune diseases in general [12,27,45–48], however, there is an open Phase IV reported on www.clincaltrials.gov for the testing of Velcade (bortezomib) in proliferative lupus nephritis. Specifically, there are two papers describing the use of bortezomib for lupus, the most recent, compares carfilzomib and ONX0914 to bortezo- mib for the treatment of lupus [12,45]. One of the major issues men- tioned in both lupus manuscripts is the potentially limited utility of chronic bortezomib administration for the treatment of lupus and other autoimmune diseases because of the dose-limiting peripheral neuropathy and cardiovascular liabilities associated with this agent. The novelty of the application of delanzomib for the treatment of lupus based upon the data presented in this manuscript is the fact that in both pre-clinical studies and phase 1 studies with delanzomib, peripheral neuropathy and cardiovascular toxicities were not observed as significant dose-limiting toxicities [49]. These facts, the oral bioavail- ability of delanzomib, and its generally manageable toxicities of delan- zomib were among the reasons for the development of this novel proteasome inhibitor as it clearly differentiated from bortezomib in these respects. These considerations are directly relevant for both the novelty and the potential utility of chronic delanzomib administration for the management of lupus and lupus nephritis based upon the exten- sive body of pre-clinical data presented in this manuscript.
In conclusion proteasome inhibition using delanzomib reduced the levels of pathogenic antinuclear antibodies and disease promot- ing inflammatory cytokines in two preclinical mouse models of SLE. In addition, delanzomib treatment of lupus nephritic mice extended survival and reduced several renal and pulmonary manifestations as- sociated with late stage lupus including the improvement of urine protein levels consistent with improved renal function. These data suggests that delanzomib may be a suitable therapy for patients suf- fering from lupus nephritis either as a monotherapy or in combina- tion with current standards of care.
Supplementary materials related to this article can be found on- line at doi:10.1016/j.intimp.2011.11.019.
Conflict of interest statement
All authors on this manuscript are employees of Cephalon, Inc.
Acknowledgments
We would like to thank the following individuals that helped make this manuscript possible: Russell Delgiacco at the Wistar Insti- tute for histology work, Julie Engiles at GetAPath, LLC., for pathology scoring and image results, Donna Bozyczko-Coyne and Denise D’An- drea for editorial help in early manuscript versions, Lisa Aimone, Kelli Zeigler and Damaris Rolon-Steele for PK help and Piyush Patel and Richard Nicholson for formulation help.
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