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Original Paper

Pharmacology 2011;88:100–113 DOI: 10.1159/000330067

Anti-Inflammatory and Immunomodulatory Effects of Bortezomib in Various in vivo Models

David Tung   a Peter H. Cheung   a Pali Kaur   b Oded Foreman   b Anoop Kavirayani   b Heather S. Hain   c Saurabh Saha   a  

a   BioMed Valley Discoveries, Kansas City, Mo. , b   Jackson Laboratory, Sacramento, Calif. , and c   Melior Discovery, Inc., Exton, Pa. , USA

tolerability was investigated in a topical imiquimod (IMQ)-induced psoriasis model. Daily application of IMQ on mouse skin induced inflamed scaly skin lesions resembling plaque-type psoriasis. Fatality was observed in the 1-mg/ml dose group. At 0.1 and 0.01 mg/ml, bortezomib potentiated IMQ-induced erythema, scaling, skin thickening, and causednecrotic lesions. Lower doses had no effect on the clinical observations. Histologically, bortezomib dose-dependently increased parakeratosis, hyperkeratosis, acanthosis, and in-flammatory cell infiltration. This study demonstrated that topical bortezomib is not suitable for the treatment of pso-riasis. Copyright © 2011 S. Karger AG, Basel

Introduction

The 26S proteasome is a proteolytic complex in the nucleus and cytosol of eukaryotic cells responsible for the degradation of a variety of regulatory proteins as well as redundant and misfolded proteins [1] . The 26S protea-some is composed of a 20S proteasome and two 19S regu-latory molecules. Inhibition of 26S can affect the degra-

Key Words

Bortezomib � Velcade � � Topical formulation � Inflammation  � Delayed-type hypersensitivity model � Psoriasis � Immune modulation � Imiquimod-induced psoriasis model

Abstract

Bortezomib (Velcade�) is a proteasome inhibitor that has been approved for the treatment of multiple myeloma and mantle cell lymphoma. It has been shown to inhibit the ex-pression of cell adhesion molecules, co-stimulatory mole-cules, and NF � B activation, to deplete alloreactive T lym-phocytes, and to decrease Th1 cytokine production. Theanti-inflammatory effects of bortezomib were further inves-tigated in this current set of studies. Systemic treatment with bortezomib was efficacious in the thioglycolate-in-duced MCP-1 production model, and the dinitrofluoroben-zene-induced delayed-type hypersensitivity model. Psoria-sis is an autoimmune disease that affects about 2% of the world population. Many treatments have been reported with varying degrees of efficacy. A topical bortezomib for-mulation was developed to minimize systemic exposure. Its

Received: February 14, 2011 Accepted after revision: May 19, 2011 Published online: August 25, 2011

David Tung, PhD BioMed Valley Discoveries 4520 Main Street, Suite 1650 Kansas City, MO 64111 (USA) Tel. +1 816 960 4625, E-Mail dtung   @   biomed-valley.com

© 2011 S. Karger AG, Basel0031–7012/11/0882–0100$38.00/0

Accessible online at:www.karger.com/pha

Anti-Inflammatory and Immuno-modulatory Effects of Bortezomib

Pharmacology 2011;88:100–113 101

dation of poly-ubiquitinated proteins. It is estimated that over 80% of all cellular proteins are processed by the 26S proteasome complex including inhibitor protein kappa B (I � B) and nuclear factor kappa B (NF � B). The role of NF � B and I � B in oncogenesis and inflammation is well established [2–7] .

The ubiquitin proteasome system can mediate inflam-mation and autoimmune diseases via multiple pathways including antigen presentation, cytokine production and cell cycle regulation [8] . It is involved in the processing of antigenic peptides presented by the MHC I molecules [9, 10] . This process is enhanced by interferon gamma (IFN- � ) which stimulates an inducible proteolytic cascade con-sisting of an activator of the 20S proteasome called PA28 and other inducible proteasome subunits [11] . The ubiq-uitin proteasome system is also involved in T-cell signal-ing. Cbl-b is a ubiquitin ligase that acts as a negative reg-ulator in T-cell receptor and CD28 signaling [12, 13] . CD28 co-stimulation results in the ubiquitination and degradation of Cbl- � by 26S proteasome, which leads to the production of proinflammatory cytokines. 26S pro-teasome is also involved in the activation of NF � B by de-grading the inhibitory protein I � B. Therefore, there ex-ists a strong rationale to target the 26S proteasome system for inflammatory and autoimmune diseases.

Bortezomib is a selective and reversible inhibitor of the 26S proteasome. It is a FDA-approved treatment for mul-tiple myeloma and mantle cell lymphoma. There exists evidence that bortezomib has anti-inflammatory effects in models of arthritis, multiple sclerosis, and graft-ver-sus-host disease [14–19] . Most of these effects were attrib-uted to NF � B inhibition by bortezomib. The ability of bortezomib to inhibit proliferation and cause apoptosis in activated T cells might also be a contributing factor, since T cells with a higher level of NF � B activation were found to be more sensitive to bortezomib treatment. Fur-thermore, bortezomib-treated CD4+ T cells have a lower level of IFN- � and IL-2 production [20] . In a rat model of streptococcal cell wall-induced chronic arthritis, borte-zomib downregulated ankle swelling, cartilage degrada-tion, and inflammatory cell infiltration. It also inhibited serum IL-6 and nitrate/nitrite levels, but failed to signifi-cantly decrease the serum IL-1 level. Peptidoglycan/poly-saccharide-induced liver lesions and liver inducible nitric oxide synthase and vascular cell adhesion molecule 1 (VCAM-1) mRNA expression were also inhibited. The level of I � B � protein in the liver was also found to be de-creased by bortezomib treatment [14] .

Psoriasis is an inflammatory disease with a strong T-cell component. The involvement of NF � B in this disease

is well established [21] . In a Hu-SCID xenograft model, the proteasome inhibitor PS-519 reduced the size, epider-mal thickness, and keratinocyte proliferation, and leuko-cyte infiltration of psoriatic lesions in the human skin graft. It also resulted in reduced 20S proteasome activity in the blood of the treated animals [22] . These observa-tions provided a strong rationale to explore the use of bortezomib in the treatment of psoriasis. However, the toxicological profile of bortezomib makes chronic sys-temic dosing undesirable [23] . Therefore, a topical formu-lation of bortezomib was developed with the goal of min-imizing systemic exposure. The tolerability of this for-mulation was examined on the skins of animals with psoriatic phenotype.

In this set of studies, the anti-inflammatory and im-munomodulatory effects of bortezomib were evaluated in various in vivo models. The development and tolera-bility testing of a topical bortezomib formulation in an imiquimod (IMQ)-induced psoriasis model will also be discussed.

Method

The anti-inflammatory and immunomodulatory effects of bortezomib were studied in a thioglycolate-induced peritonitis model and a 4-dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) model. In order to facilitate long-term dosing and minimize systemic toxicity, a topical formula-tion of bortezomib was developed and the tolerability was evalu-ated in an IMQ-induced psoriasis model. All in vivo protocols were approved by the IACUC of the Jackson Laboratory, and the IACUC of Melior Discoveries, Inc.

Thioglycolate-Induced Peritonitis Model Monocyte infiltration into a site of injury or irritation is an

integral part of the inflammatory process. Thioglycolate-induced MCP-1 production can be used as a biomarker for inflammatory response [24] . Male Aai:ICR BR mice (S.A. Ace Animals, Boyer-town, Pa., USA) at 8–10 weeks of age were used for this study. The animals were housed 6/cage in individually ventilated polycar-bonate cages with HEPA-filtered air and kept on a 12-hour light dark cycle. Standard rodent diet and water were provided ad libi-tum. 3% thioglycolate broth was prepared by dissolving 3 g of thioglycolate (Difco, Houston, Tex., USA) in 100 ml of boiling water. The mice received 1 ml of room-temperature thioglycolate intraperitoneally. Bortezomib (1.5 mg/kg) or dexamethasone (20 mg/kg) was administered intraperitoneally 15 min prior to thio-glycolate injection. Two hours after challenge, peritoneal lavage fluid was collected. It was spun at 200 rpm for 5 min and the su-pernatant was used for analysis of the MCP-1 level by ELISA (R&D Systems, Minneapolis, Minn., USA).

DNFB-Induced DTH Model 7-week-old female BALB/cJ mice (Jackson Laboratory, West

Sacramento, Calif., USA) were housed in individually and posi-

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Pharmacology 2011;88:100–113102

tively ventilated polycarbonate cages with HEPA-filtered air at a density of 2 mice/cage. The animal room was lighted with artifi-cial fluorescent lighting, with a controlled 12-hour light/dark cy-cle. The normal temperature and relative humidity ranges in the animal rooms were 22 8 4   °   C and 50 8 15%, respectively. Filtered tap water, acidified to a pH of 2.8–3.2, and LabDiet 5LL4 were provided ad libitum. The study was conducted in an AAALAC-accredited facility and was approved by the Jackson Laboratory Animal Care and Use Committee following all NIH guidelines.

The anesthetized animals were sensitized to DNFB by topical application of a 0.5% DNFB solution in acetone/olive oil (4: 1) to their shaved backs for 2 consecutive days [25] . Five days after sen-sitization, 0.2% DNFB was applied to the dorsal surface of the right ear. The left ear was painted with acetone. Once daily intra-peritoneal injection of bortezomib (0.17 and 1.5 mg/kg) or 2% hydrocortisone was administered at 1 h prior to challenge, and 23 and 47 h after challenge. Ear thickness was measured using a mi-crometer at baseline, the time of sensitization, and 24, 48 and 72 h after challenge. Three measurements of ear thickness for each mouse were taken at all time points. Four mice from each group were sacrificed at 24, 48 and 72 h after dose. A terminal blood sample was collected and processed for a cytokine profile and complete blood count. Blood samples for CBC were collected in lithium heparin tubes. Cytokine analysis was carried out using the Luminex 100 system (Luminex Corporation, Austin, Tex., USA). Complete blood count analysis was performed only at the 24- and 72-h time points. Weekly body weights and clinical ob-servations were monitored.

IMQ-Induced Psoriasis Model IMQ is a TLR7/8 ligand and a potent immune activator. Daily

application of IMQ on mouse skin induced inflamed scaly skin lesions resembling plaque-type psoriasis with increased epider-mal proliferation, abnormal differentiation, and epidermal accu-mulation of inflammatory cells [26] .

Eight-week-old BALB/cJ mice (Jackson Laboratory, Sacramen-to, Calif., USA) were housed in individually and positively venti-lated polycarbonate cages with HEPA-filtered air. Husbandry con-ditions were as stated above. Animals were randomized into groups of 8. All mice received daily topical application of 62.5 mg IMQ cream (5%) on the shaved back and the right ear for 6 con-secutive days. Control animals received petroleum jelly on the shaved back and right ear. Animals in the treatment groups re-ceived topical treatment of bortezomib or topical 0.05% clobetasol (Altana, N.Y., USA) starting from the 2nd day of IMQ application. The concentration of the topical bortezomib formulation was be-tween 1 mg/ml and 0.1 � g/ml. Treatment continued for a total of 9 days. The animals were scored independently for clinical param-eters of disease based on the degree of erythema, scaling and thick-ening on a scale of 0–4, with 4 being the most severe. In general, a score of 0 represents no observable pathology. A score of 1–4 cor-responds to 0–25, 25–50, 50–75, and 75–100% of the shaved area affected, respectively, with increasing severity. Clinical observa-tions and ear thickness of the right ear were recorded on days 0, 2, 4, 6, and 8. Body weights and general clinical observations were recorded daily. There were 8 animals/group. Due to the size of the studies, the full dose range was studied in 3 different sets of ex-periments, examining bortezomib concentrations of 1, 0.1 and 0.01 mg/ml, and 1 and 0. 1 � g/ml, respectively. Eight animals were used in each group for clinical and histological examinations.

Tissues After the mice were euthanized, sections of dorsal skin and ear

pinna were harvested, fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned at a thickness of 5 � m, and stained with hematoxylin and eosin. Spleen samples were also harvested and weighed.

Histological Scoring The skin sections were scored by a board-certified veterinary

pathologist without prior knowledge of the treatment groups. Each sample was scored on 7 individual parameters: parakerato-sis, hyperkeratosis, acanthosis, epidermal micro-abscesses, basil-lar papillae, dermal inflammatory infiltrates, and dilated tortu-ous capillaries. Each parameter was graded according to a 4-point severity score ( table 1 ).

Development of a Topical Formulation of Bortezomib Commercially available Velcade � lyophilisate (bortezomib;

Janssen-Cilag AG, Germany) was dissolved in 0.9% sodium chlo-ride solution prior to use. Our internal data indicated that hy-droxypropyl cellulose (HPC) was chemically and physically com-patible with bortezomib with good stability over a 2-week period. A formulation containing 70.8% propylene glycol (PG), 28.2% wa-ter and 1.0% HPC w/v was selected as a vehicle to dissolve the Velcade lyophilisate. All materials used in this study are endo-toxin free.

HPLC Method Conditions An Agilent 1100/1200 Series HPLC was used for the analysis

of test article stability. The eluent was a 50-mmol/l methanol/wa-ter/phosphate buffer (50: 45: 5 v/v/v) at pH 2. The samples were passed through a 25   °   C Agilent Zorbax SB-C18 column at a flow rate of 1.0 ml/min for 50 min. The wavelength of detection was set at 253 nm, and the diluent was methanol/water (2: 1 v/v).

Stability Testing of Bortezomib in HPC Formulation Bortezomib was dissolved at a concentration of 1 mg/ml in

70.8% PG, 28.2% water and 1.0% HPC w/v solution, by adding the vehicle to the bortezomib and mixing on a vortex mixer for ap-proximately 2 min at room temperature until a clear formulation was obtained. The formulation was then analyzed at 0, 6, and 24 h at room temperature by HPLC for bortezomib content. Bor-tezomib was also dissolved in methanol/water (2: 1 v/v) at 1 mg/ml (as the sample preparation solution for HPLC determination).

Stability Testing of Bortezomib after Freeze-Thaw Cycle Two formulations of 0.1 and 1 mg/ml bortezomib in 70.8% PG,

28.2% water and 1.0% HPC w/v were frozen at –18   °   C for 16 h. The frozen formulations were removed and left at room temperature to thaw and equilibrate for 24 h, examined visually for any pre-cipitation, and analyzed by HPLC.

Data Analysis and Statistics All data are presented as mean 8 SEM. One-way analysis of

variance (ANOVA) and non-parametric Mann-Whitney U test were used to analyze statistical significance. Analysis of the data was performed using MS Excel (Microsoft Inc., San Jose, Calif., USA).

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Pharmacology 2011;88:100–113 103

Results

Effect of Bortezomib Treatment on the Thioglycolate-Induced Peritonitis Model MCP-1 is a CC chemokine with potent monocyte che-

motactic activity [27, 28] via its interaction with the G protein-coupled receptor CCR2 [29–31] . It plays a critical role in the recruitment of several leukocyte populations including monocytes, T lymphocytes, and NK cells [32] . Its role has been established in many inflammatory dis-eases, including atherosclerosis, pulmonary fibrosis, and multiple sclerosis [33] . MCP-1 is produced by a variety of cell types, including macrophages, fibroblasts, epithelial cells, and endothelial cells. It had been shown to be the key chemotactic factor in thioglycolate-induced macro-phage chemotaxis in vivo [34, 35] .

In the current model of thioglycolate-induced perito-nitis, MCP-1 was used as a biomarker for the severity of the inflammatory response. Intraperitoneal injection of a 3% thioglycolate broth resulted in the production of 2,970.27 pg/ml of MCP-1 in the peritoneal lavage fluid. Systemic treatment with bortezomib significantly re-duced the MCP-1 production to 841.83 pg/ml ( fig. 1 ).

Table 1. T he psoriatic score rating scale

Grade and score

Parakeratosis Keratin layerhyperkeratosis

Epidermal acanthosis

Epidermal microabscesses

Basillar papillae Dermal inflammatory infiltrates

Dilated tortuous capillaries

None (0) No visible fociof keratinocytenuclei over theepidermal surface

No increase in keratin layerthickness

No foci of hyper-plasia of the squamous celllayer

No foci of neutrophils and lymphocytes aggregation

No foci of rete ridge-like papillary extensions of the hyperplastic epidermis into the dermal stroma

No foci of inflammatory cell aggregatesin the dermis

No foci of dilated tortuous capillaries

Minimal (1) Rare foci <15%increase in thickness

Rare foci with slight to prominent thickening

Rare foci Rare foci Rare foci Rare foci

Mild (2) A few foci 15–30% increasein thickness

A few foci with prominent thickening

A few foci A few foci A few foci A few foci

Moderate (3) Multiple foci 30–60% increasein thickness

Multiple foci with prominentthickening

Multiple foci Multiple foci Multiple foci Multiple foci

Severe (4) Extensive foci involving >70% of the epidermal surface

>60% increase in thicknessinvolving >70% of the epidermalsurface

Extensive foci involving >70% of the epidermal surface with marked thickening

Numerous foci involving >70% of the epidermal surface

Numerous foci involving >70% of the dermis

Extensive foci involving >70%of the dermis

Numerous foci involving >70% of the dermis

The histological sections from the skin samples of the animals were scored on seven histological parameters according to this scale.

4,000

500

0

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3,500

VehicleNaïve

*

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-1 le

vel (

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mib

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ne

*

Fig. 1. Bortezomib treatment significantly reduced MCP-1 pro-duction in a thioglycollate-induced peritonitis model. The level of MCP-1 production induced by thioglycolate is shown. 1.5 mg/kg bortezomib administered intraperitoneally 15 min before thio-glycolate challenge significantly reduced the MCP-1 level in the peritoneal lavage fluid 2 h after challenge. *  p ̂ 0.05 as compared to vehicle.

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Pharmacology 2011;88:100–113104

Effect of Bortezomib Treatment on DNFB-Induced DTH Model It is well documented that repeated topical applica-

tions of DNFB to the ears of BALB/c mice can result in DTH [36–38] . Differential expression of IL-2 and IFN- � mRNAs was found in the challenged ears. IL-4 and IL-5 mRNAs in the lymph nodes were detected. IL-12 and IFN- � were identified as the major cytokines instrumen-tal for the DTH response [39–42] . Due to the many as-pects of the immune components involved, the DNFB DTH model is ideal for analyzing the immune-modula-tory effects of bortezomib.

In this study, bortezomib was administered at 1 h pri-or to challenge, and 23 and 47 h after challenge. Ear thick-ness increase was significantly inhibited at the 1.5-mg/kg dose ( fig.  2 ), but there was no effect at the 0.17-mg/kg dose. Some animals in the high-dose group were begin-ning to show signs of moribund after the dosing period. Thus, long-term systemic dosing with bortezomib would not be feasible.

Effect of Bortezomib Treatment on Blood Cell Population in the DTH Model Blood samples were collected from selected animals at

24, 48, and 72 h after the beginning of the bortezomib dosing paradigm. CBC analysis was performed on blood samples collected at 24 and 72 h. The number of platelets,

neutrophils, lymphocytes, monocytes, eosinophils and basophils were measured. DNFB challenge led to a sig-nificant increase in the number of platelets, neutrophils and monocytes in the serum ( fig. 3 a). The eosinophil lev-el was also increased, but not significantly different from the level in the naïve animals. Hydrocortisone treatment significantly attenuated the increase of neutrophils, monocytes and eosinophils.

24 hours after dosing, systemic treatment with both 0.17 and 1.5 mg/kg bortezomib significantly decreased the number of monocytes in the serum, but did not im-pact the number of platelets, lymphocytes, eosinophils or basophils.

72 h after DNFB challenge, the level of neutrophils in the blood sample of the vehicle-treated animals was still significantly elevated over the naïve animals. 2% hydro-cortisone significantly downregulated the concentration of lymphocyte even as compared to the naïve animals, and cannot be considered as physiologically relevant. Sys-temic treatment with either 0.17 or 1.5 mg/kg bortezomib did not decrease the number of WBC or platelets at this time point.

Effect of Bortezomib Treatment on Serum Cytokine in the DTH Model Terminal blood samples from the animals were col-

lected at 24, 48 and 72 h after systemic bortezomib treat-

0.150 1 2 3 4 5 6 7 8

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029

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)

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Treatment was administered 1 h beforechallenge and 23 and 47 h after challenge

Topical DNFB sensitization DNFB challenge

*

*

*

*

*

*

*

*

NaïveVehicleHydrocortisone0.17 mg/kg bortezomib1.5 mg/kg bortezomib

Fig. 2. Systemic treatment with bortezo-mib at 1.5 mg/kg significantly inhibited ear swelling in the DNFB-induced DTH model. The animals were treated with bortezomib 1 h before challenge, and 23 and 47 h after challenge just before the ear thickness measurements were taken. 0.17 mg/kg bortezomib has no effect on ear swelling caused by DNFB challenge. *  p ̂ 0.05 as compared to vehicle.

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Pharmacology 2011;88:100–113 105

ment. The serum levels of IFN- � , MCP-1, RANTES and IL-12 were analyzed.

The serum level of IFN- � was significantly elevated from the level of the naïve animals 24 h after DNFB chal-lenge. The IFN- � level was downregulated by hydrocor-tisone, but not by bortezomib treatment ( fig. 4 a). The se-rum IFN- � returned to the baseline level of the naïve an-imals 48 and 72 h after challenge.

DNFB challenge led to elevation in the MCP-1 level in serum. This was downregulated by hydrocortisone treat-ment, but not bortezomib. The MCP-1 level in vehicle-

treated animals decreased at 48 h. Both hydrocortisone and bortezomib did not downregulate the MCP-1 level at this time point. 72 h after challenge, only hydrocortisone treatment significantly attenuated MCP-1 production in the serum ( fig. 4 b).

The RANTES level was not significantly elevated by DNFB challenge. Both hydrocortisone and bortezomib did not significantly affect the serum RANTES level at all time points ( fig. 4 c).

The serum IL-12 level in the vehicle-treated DNFB-challenged animals slightly increased above the level of

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NaïveVehicleHydrocortisone0.17 mg/kg bortezomib1.5 mg/kg bortezomib

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Monocytes Eosinophils Basophils

Fig. 3. The effect of bortezomib treatment on the level of various cell populations in the circulation 24 and 72 h after dose. The cel-lular levels in blood 24 and 72 h after challenge are shown. a 24 h after challenge, bortezomib significantly decreased the number of monocytes in the blood sample, but it had no effect on the oth-

er cell populations. b 72 h after challenge, bortezomib treatment did not impact the level of circulating blood cells. Besides neutro-phils, all other cell populations returned to the baseline level. Hy-drocortisone decreased the level of lymphocytes to below that of the naïve animals. *  p ! 0.05 compared to vehicle control.

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Pharmacology 2011;88:100–113106

naïve animals 24 h after challenge. Treatment with hy-drocortisone or bortezomib did not affect the serum IL-12 level at this time point. The levels of IL-12 at later time points were also not affected by bortezomib or hydrocor-tisone treatment.

Stability of the Topical Bortezomib PG/HPC Formulation In order to support the potential of long-term dosing,

a topical formulation of bortezomib was developed to minimize systemic exposure. Several variations of the current formulation were evaluated, but a formulation of 70.8% PG, 28.2% water and 1.0% HPC w/v solution was selected due to its ease of synthesis and stability. The

Velcade lyophilisate dissolved easily at 1 mg/ml in this formulation. The bortezomib PG/HPC formulation was a clear colorless viscous gel. This appearance did not change over the observation period. The resulting solu-tion was physically stable for 24 h at room temperature and showed a slight increase of 0.61 area% ( table 2 a).

After the freeze-thaw cycles, there was no change in the physical appearance of the formulation. The stability characteristics of the 1-mg/ml bortezomib (Velcade) PG/HPC solution after cooling to –18   °   C and warming to room temperature for 24 h are provided in table 2 b.

Bortezomib in PG/HPC solution was chemically sta-ble after freeze-thawing and storage at room temperature for 24 h. The percent degradation increased from 0.59%

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Fig. 4. The effect of bortezomib treatment on serum IFN- � , MCP-1, RANTES, and IL-12 levels. a The IFN- � level was transiently elevated 24 h after challenge, and returned to baseline by 48 h. *  p ! 0.05 compared to vehicle control at 24 h. Hydrocortisone significantly inhibited this transient increase. Bortezomib treat-ment had no effect. b The MCP-1 expression in the serum was elevated 24 h after challenge. This elevation was attenuated by hydrocortisone treatment, but not by bortezomib. *  p ! 0.05 com-

pared to vehicle control at 24 h; #  p ! 0.05 compared to vehicle control at 72 h. c The serum RANTES level was not significantly upregulated in this model. The slight increase in the animals treated with hydrocortisone at 24 h, and with bortizomib at 72 h was not statistically significant. d The serum IL-12 level was not significantly elevated after challenge. There was no significant ef-fect observed in any of the treatment groups.

Anti-Inflammatory and Immuno-modulatory Effects of Bortezomib

Pharmacology 2011;88:100–113 107

at 0 to 0.96% at 24 h. However, further diluted 0.1-mg/ml bortezomib in PG/HPC formulation showed increased degradation compared to the 1.0-mg/ml bortezomib for-mulation over the same time period. A further 9.5% deg-radation was observed indicating chemical instability of bortezomib in the presence of excess PG/HPC under short-term storage conditions at room temperature. For this reason, the dosing formulation was prepared imme-diately before dosing.

Effect and Tolerability of Bortezomib Treatment in Naïve Animals and Animals with Psoriatic Phenotype Induced by IMQ The tolerability of the PG/HPC vehicle cream and the

same cream loaded with 1 and 0.1 mg/ml bortezomib was tested in naïve animals. Within 3 days of dosing, 3 ani-mals in the high-dose group died, and the rest became moribund. Based on the IACUC recommendation the re-maining animals were sacrificed. The animals in the ve-hicle and 0.1-mg/ml groups appeared normal with no ob-servable clinical signs of irritation. When these animals were sacrificed 5 days later, there was no observable his-topathology in the skin samples (data not shown). In sub-sequent tolerability studies performed on psoriatic skin, bortezomib concentrations of 0.1 mg/ml and lower were used.

Repeated topical application of IMQ on mouse skin induced plaque-type psoriasis-like skin lesions. Clinical observations in the form of erythema, scaling, and skin thickening began to manifest after 2 days of IMQ chal-lenge. Topical clobetasol ameliorated these clinical obser-vations ( fig. 5 ).

Skin erythema became observable after 2 days of IMQ challenge, which is reflected in an elevation in the ery-thema score. Bortezomib at a concentration of 0.1 mg/ml potentiated skin redness after 3 days of application ( fig. 5 a). This concentration was shown not to cause any pathology in naïve animals in a previous safety study. However, at concentrations of ! 0.01 mg/ml, the bortezo-mib formulation did not have any impact on the erythe-ma (data not shown).

Skin dryness is part of the pathological condition in psoriasis that often progresses to scaling. Bortezomib sig-nificantly potentiated skin scaling at 0.1 and 0.01 mg/ml after 3 days of treatment ( fig. 5 b), but doses at lower con-centrations did not have any effect (data not shown).

In this model, skin thickness increase is an observable clinical parameter ( fig. 5 c). However, the extent of this pathology is quite mild. 0.1-mg/ml bortezomib treatment significantly potentiated this pathology as early as 2 days

after treatment. This was not observed at lower concen-trations of bortezomib. To further examine this skin-thickening phenomenon, IMQ was also applied to the right ears of the animals, and the observed tissue also re-ceived bortezomib and clobetasol. The ear thickness in-crease was completely inhibited by clobetasol. Bortezo-mib treatment at all concentrations did not have an effect ( fig. 5 d).

Upon completion of the experiment, the animals were sacrificed, and dorsal skin samples were harvested for histological analysis. The histological features of para-keratosis, acanthosis and dermal inflammation are illus-trated in figure 6 . IMQ challenge resulted in a major in-crease in parakeratosis, hyperkeratosis, acanthosis, and dermal inflammatory infiltrate.

Parakeratosis, defined as accumulation of immature keratinocytes in the epidermal layer, was potentiated at the two highest concentrations of bortezomib ( fig.  7 a).

Table 2. In vitro analysis of bortezomib stability.a 1 mg/ml bortezomib (Velcade) was stable in PG/HPC solution after 24 h at room temperature

Time pointh

Total degra-dation, %

0 0.596 0.94

24 1.20

The samples were analyzed using HPLC as detailed in the Methods section. The maximum total degradation observed at 24 h was 1.2%.

b Th e eff ect of a freeze-thaw cycle on the stability of the borte-zomib formulation was examined

Time point Total degra-dation, %

Concentration of formulation: 1 mg/mlTime 0, before freezing 0.59After 24 h of thawing at room temperature 0.96

Concentration of formulation: 0.1 mg/mlTime 0, before freezing 0.59After 24 h of thawing at room temperature 10.09

The formulations at 0.1 and 1 mg/ml were frozen at –18° C for 16 h, then thawed at room temperature. The 1-mg/ml formulation was stable, but there was 10.09% degradation in the 0.1-mg/ml sample. The stability of the formulation is concentration depen-dent.

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Pharmacology 2011;88:100–113108

Treatment with lower concentrations was well tolerated with the skin sample showing the same degree of para-keratosis as the vehicle control group ( fig. 7 b). There is a slight variation in the score of the naïve and vehicle ani-mals between the 2 experiments. This is attributed to the experimental variability.

Hyperplasia of keratinocytes leads to increased thick-ness of the epidermal keratin layer (hyperkeratosis). None of the treatment group had any positive effect on hyper-keratosis ( fig.  7 b). However, the two higher concentra-tions of bortezomib at 0.1 and 0.01 mg/ml dramatically augmented the hyperkeratosis ( fig. 7 a).

Squamous cell hyperplasia in the epidermis (acantho-sis) is another factor that contributes to the thickening of psoriatic skin. Acanthosis was often evident in the epi-dermis of the IMQ-challenged animals showing multiple foci with prominent thickening. Concentrations of bor-tezomib at 0.1 and 0.01 mg/ml, but not lower concentra-tions, greatly potentiated this effect ( fig. 7 ).

Dermal inflammatory cell infiltration was observed in the IMQ-challenged animals. This observation was significantly inhibited by clobetasol, but exacerbated by 0.1- and 0.01-mg/ml concentrations of the bortezomib formulation.

00 2

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Fig. 5. Effect of bortezomib treatment at 0.1 and 0.01 mg/ml on various clinical parameters. a IMQ challenge increased the ery-thema of the treated skin. This was significantly inhibited by clo-betasol after 2 days of treatment. 0.1 mg/ml bortezomib signifi-cantly potentiated the erythema score, but lower concentrations had no effect. b Dryness-related scaling of the skin was observable after 4 days of IMQ challenge. 0.1 and 0.01 mg/ml bortezomib treatment dose-dependently increased the scaling score, but there

was no potentiating effect at lower doses (data not shown). c Mild increase in skin thickness was observed after IMQ challenge. 0.1 mg/ml bortezomib significantly potentiated this clinical observa-tion, but the lower concentrations of bortezomib had no effects. d IMQ applied to the right ears of the animals resulted in thicken-ing of the skin. Clobetasol significantly inhibited this increase. Bortezomib at all concentrations had no effect on this observation. *  p ! 0.05 as compared to vehicle control at the same time point.

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Pharmacology 2011;88:100–113 109

Epidermal micro-abscesses, as defined by foci of dis-crete aggregates of neutrophils and lymphocytes within the squamous cell layer, are not a common feature in this model. However, it was observed in the vehicle-treated an-imals and the 0.1-mg/ml bortezomib-treated animals. Foci of rete ridge-like papillary extensions of the hyperplastic epidermis into the dermal stroma are another rare feature

of this model. Its occurrence is limited to rare foci. In ani-mals treated with 0.1 and 0.01 mg/ml bortezomib, several such foci were observed in the histological sections ( fig. 7 ).

Dilated tortuous capillaries are generally not found in the skin of IMQ-challenged animals. However, bortezo-mib at a concentration of 0.1 and 0.01 mg/ml resulted in the appearance of rare foci of such anomaly.

a b

c d

e f

g h

Fig. 6. Histological features of the psori-atic lesions. A In naïve animals, psoriatic features are not present in skin. Parakera-tosis, acanthosis and inflammatory infil-trates are not detectable. B In vehicle-treat-ed animals, the epidermis is covered by a serocellular crust (Sc), acanthosis (Ac) and dermal inflammation (short arrow) were common. In skin sections of IMQ-chal-lenged animals treated with bortezomib at 0.0001 mg/ml ( C ), 0.001 mg/ml ( D ), 0.01 mg/ml ( E ), and 0.1 mg/ml ( F ), psoriatic fea-tures such as parakeratosis (long arrow), acanthosis (Ac) and dermal inflammation (short arrow) were evident. G , H Histolog-ical features of the clobetasol-treated ani-mals. Acanthosis and hyperkeratosis with mild parakeratosis was observed. Inflam-matory infiltration was also mild. HE. Magnification: 100 ! ( A–E , G) , and 200 ! ( F , H ).

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Pharmacology 2011;88:100–113110

Discussion

The proteasomal system had been implicated in many inflammatory and autoimmune diseases. Levels of circu-lating proteasomes in serum samples from patients with rheumatoid arthritis, SLE, myositis, and Sjögren’s syn-drome correlated with disease severity [43, 44] . Protea-some inhibition has been shown to be effective in modu-lating macrophages’ inflammatory gene expression in ro-dent models of shock [45] . In a model of peritonitis, we demonstrated that bortezomib successfully downregu-lated systemic MCP-1 production. MCP-1 (CCL2) is a CC chemokine that recruits monocytes, memory T cells, and dendritic cells [46, 47] . This 13-kDa monomeric polypep-

tide is primarily secreted by monocytes, macrophages and dendritic cells. It is strongly chemotactic for mono-cytes and basophils, and causes basophil and mast cell degranulation [48–50] . MCP-1 expression has been shown to be linked to NF � B activation. Since bortezomib had been shown to downregulate NF � B activation, it stands to reason that it should affect MCP-1 expression as well. The inhibitory effect of bortezomib on MCP-1 production in this thioglycolate-induced peritonitis model is in line with the rationale and findings in the lit-erature. This set of data prompted further exploration of bortezomib’s immunomodulatory effects in the DTH model.

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NaïveVehicleClobetasol0.001 mg/ml bortezomib0.0001 mg/ml bortezomib

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*

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*

Fig. 7. Histological score of various parameters in the psoriatic le-sions. a Bortezomib at 0.1 and 0.01 mg/ml potentiated the psoria-sis phenotype as evident in the histologic score for parakeratosis, hyperkeratosis, acanthosis, and inflammatory infiltrates. Epider-mal micro-abscesses, rete ridge-like papillary extensions of the hyperplastic epidermis into the dermal stroma, and dilated capil-

laries were not common features, but these pathologies were ag-gravated by the two highest doses of bortezomib. b Bortezomib at concentrations below 0.001 mg/ml did not have any effect on the pathological features of the psoriatic skin samples. Epidermal mi-cro-abscesses, rete ridge infiltration into the dermal layer, and dilated capillaries were not seen in any of the treatment groups.

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Pharmacology 2011;88:100–113 111

DTH is an autoimmune response with a well-estab-lished T-cell component. The role of the proteasome sys-tem was demonstrated by a selective proteasome inhibi-tor, epoxomicin, which prevented I � B � degradation in-duced by TNF- � in HeLa cells, and inhibited pic ryl-chloride-induced contact sensitivity in mice [51] . In the current study, bortezomib successfully downregulated ear swelling upon antigen challenge. It is generally be-lieved that the anti-inflammatory effect of bortezomib is due to myeloablation. In the current experiment, bor-tezomib decreased the number of monocytes in the se-rum within 24 h after the first treatment without impact-ing the absolute number of other cell types. Even though we did not examine the local infiltration of inflamma-tory cells, this issue was previously addressed by Yanaba et al. [52] , who observed that the bortezomib-inhibited DTH response was associated with a decrease in inflam-matory cell infiltration in the challenged skin [52] .

In order to further elucidate the immunomodulatory effects of bortezomib, the systemic levels of MCP-1, RANTES, IFN- � , and IL-12 at 24, 48 and 72 hours after DTH challenge were examined. The maximum elevation of MCP-1 were observed 24 h after challenge. Our previ-ous data showed that bortezomib was successful in down-regulating MCP-1 production in the thioglycolate-in-duced peritonitis model. However, it did not significantly downregulate the MCP-1 level at both dose levels in this DNFB-induced DTH model, though there is a trend to a lower level of MCP-1 production in all the bortezomib-treated animals. 48 and 72 h after challenge, the serum MCP-1 level in the DNFB-challenged decreased, and the pharmacological window became too small to demon-strate any inhibitory effect of bortezomib.

RANTES (CCL5) is a CC chemokine that is chemotac-tic for T cells, eosinophils, and basophils. It works syner-gistically with IL-2 and IFN- � that are released by T cells. The systemic RANTES level was not significantly elevat-ed in this model. Even though hydrocortisone treatment at 24 h and 0.17-mg/kg bortezomib treatment at 72 h showed a trend to increasing the RANTES level, the ele-vation was not significantly above baseline. RANTES is usually expressed 3–5 days after T-cell activation. It is possible that the time course of this model was too short for RANTES to play a major role.

IFN- � is produced mainly by Th1 cells, NK cells and CD8+ cytotoxic T lymphocytes [53] . Its immunomodula-tory effects stem from its ability to potentiate antigen pre-sentation and lysosomal activity of macrophages, in-crease expression of class I MHC molecules, promote leu-kocyte migration, increase NK cell activity, and activate

inducible nitric oxide synthase [54] . DNFB challenge led to a significant increase in serum IFN- � level 24 h after challenge which subsequently returned to baseline level after this spike. This is consistent with a typical DTH re-sponse. Hydrocortisone treatment was the only thera-peutic that significantly downregulated this increase.

During antigenic stimulation, IL-12 is produced by dendritic cells [55] , macrophages and B-lymphoblastoid cells. It is involved in the differentiation of naïve Th0 cells into Th1 cells, and can stimulate the production of IFN- � and TNF- � from T and NK cells. IL-12 also potentiates the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. In the DNFB-induced DTH model, even though the serum IL-12 level in the vehicle-treated ani-mals rose above the baseline level of the naïve animals, the magnitude of the increase was not significantly dif-ferent. None of the therapeutic treatments significantly impacted this increase during the 72-hour observation period. It is possible that the mild increase in IFN- � level did not provide a large enough pharmacological window to discern a treatment effect.

The toxicity of bortezomib makes chronic systemic dosing undesirable. Therefore, a novel bortezomib formu-lation containing 70.8% PG, 28.2% water and 1.0% HPC w/v was specifically developed to support topical applica-tion for skin diseases such as psoriasis. Safety studies con-firmed that the vehicle cream was well tolerated by the naïve animals (data not shown). The tolerability of the top-ical bortezomib at concentrations between 1 and 0.0001 mg/ml were evaluated on psoriatic skin phenotype in-duced by IMQ. Since the 1-mg/ml dose induced fatality in the animals within 2 days, this is a clear indication that the locally applied formulation was leading to systemic expo-sure. The 0.1-mg/ml dose of bortezomib significantly ex-acerbated erythema, scaling and skin thickening. At this dose, the estimated local concentration was 260 � mol/l. There is evidence in 4 murine and 2 human squamous cell carcinoma lines that bortezomib at 0.1 � mol/l can cause toxicity and caspase-mediated apoptosis [56] . Some head and neck squamous cell carcinoma lines even showed tox-icity at a concentration as low as 1 nmol/l [57] . The local concentration in our study was much higher. At this high concentration, off-target polypharmacology might also occur, and the mechanism is difficult to elucidate. A re-view of the literature did not uncover any prior reports of the irritating effect of bor tezomib on skin. There is also no evidence of a link between proteasome inhibition and the skin pathologies as reported here. There is evidence that continuous inhibition of the proteasome by bortezomib can lead to increased expression of proteasomal subunits

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Pharmacology 2011;88:100–113112

[58] . This might be a potential explanation for the poten-tiating effect of bortezomib on the inflammatory pheno-type observed.

In this series of studies, we confirmed the anti-inflam-matory effect of bortezomib in a thioglycolate-induced peritonitis model and a DNFB-induced DTH model. It was puzzling that despite the inhibition of ear swelling in the DTH model, there was little effect on several proin-flammatory cytokines. However, the circulating level of monocytes was inhibited, which was in line with the mechanism of action of bortezomib. When the tolerabil-ity of bortezomib was examined in psoriatic skin pheno-

type, the agonistic effects were prominent at the histo-logical level. The therapeutic index is defined by a balance between the level of toxicity and the level of efficacy. It is possible that bortezomib’s irritant effect confounded any potentially efficacious effects at the dose level tested. Even though this study shows that the toxicity of bortezo-mib makes it unsuitable for chronic application, other proteasome inhibitors with better toxicology profile are still worth further investigation. Another option to ex-plore is to decrease the duration of bortezomib exposure, and thus decrease the possibility of potentiating the in-flammatory response.

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