Combination therapy of pentoxifylline and TNFα monoclonal antibody in dextran sulphate-induced mouse colitis

Combination therapy of pentoxifylline and TNFa monoclonalantibody in dextran sulphate-induced mouse colitis

S. MURTHY, H. S. COOPER*, H. YOSHITAKE, C. MEYER, C. J . MEYER & N. S. MURTHY

Division of Gastroenterology and Hepatology, MCP Hahnemann University and *Fox Chase Cancer Center, Philadelphia, PA,

USA

Accepted for publication 26 September 1998

INTRODUCTION

Crohn's disease and ulcerative colitis are chronic

diseases of unknown origin. These diseases exhibit

periods of quiescence and relapses with many overlap-

ping clinical features and therapeutic responses. These

diseases differ fundamentally in their histological ap-

pearance and disease locations. Several recent develop-

ments have shown that in¯ammatory bowel disease is a

genetically complex multifactorial disease. The genetic

susceptibility of individuals to in¯ammatory bowel

disease is driven by environmental factors which

aggravate the immune system, resulting in the

production of several soluble mediators, overt in¯am-

mation and tissue damage. These mediators include

arachidonic acid metabolites, complement-derived

chemotactic peptides and the increased secretion of

SUMMARY

Background: Tumour necrosis factor-alpha (TNFa) has

been suspected of playing an important role in the

pathogenesis of in¯ammatory bowel diseases, and has

become a target for the treatment of these diseases.

Open-label, placebo controlled studies have shown that

engineered CDP571 and chimeric anti-TNF antibody

(cA2) provide a signi®cant bene®t in Crohn's disease.

Since these antibodies have to be used repeatedly to

maintain remission in in¯ammatory bowel disease,

there is a concern that their use may compromise host

defence and produce toxic side-effects.

Methods: We evaluated the combined use of mouse

speci®c TNFa mab (25 lg/mouse, Endogen) and pen-

toxifylline (PF, 100 mg/kg/day, p.o., TNFa release

inhibitor) in the DSS (3% dextran sulphate solution)

model of mouse colitis. Colitis was induced by the

feeding of 3% DSS for three cycles. The study groups

were: Group I: single injection of rat anti-mouse IgG,

Group II: single injection of TNFa mab, Group III: daily

PF for three cycles, Group IV: single injection of TNFamab + PF for three cycles, Group V: TNFa mab at the

beginning of each cycle (three injections) and Group VI:

TNFa mab (three injections) + daily PF for three cycles.

Daily disease activity (DAI) was measured throughout

the study. At the end of each cycle, colon tissue was

processed for histology, myeloperoxidase (MPO) and

plasma TNFa.

Results: Mice treated with a single injection of TNFaalone or TNFa mab + PF showed signi®cantly lower

DAI, in¯ammation scores and ulcer index compared

with the IgG treated group. Mice treated with TNFamab + PF had no ulcers. Multiple injections of TNFamab or TNFa mab + PF showed greater inhibition in

DAI and cytokines in the ®rst two cycles. However, in

the third cycle, multiple injections of TNFa mab showed

adverse proin¯ammatory effects.

Conclusion: The simultaneous administration of pentox-

ifylline and TNFa mab may enhance therapeutic out-

comes in in¯ammatory bowel disease and reduce the

side-effects associated with the repeated use of TNFa mab.

Correspondence to: Dr S. Murthy, Division of Gastroenterology and

Hepatology, MS 131, Hahnemann University Hospital, Broad and Vine,

Philadelphia,

PA 19102±1192,USA.E-mail: [email protected]

Aliment Pharmacol Ther 1999; 13: 251±260.

Ó 1999 Blackwell Science Ltd 251

pro-in¯ammatory cytokines such as IL-1, IL-6, IL-8,

IFNa and TNFa in in¯ammatory bowel disease.1 At the

same time, certain anti-in¯ammatory cytokines of Th2

cells, such as IL-4 and IL-10, which down-regulate the

cytokines of Th1 lymphocytes, are produced at lower

levels.2, 3 This tips the balance towards pro-in¯amma-

tory processes to cause overt in¯ammation and tissue

damage. Studies carried out in gene knockouts of IL-24

and IL-10,5 in mice, resulted in the development of

pathogen-induced colitis in these animals, suggesting a

protective role for these cytokines to an attack from

ubiquitous pathogens. Whether the altered production

of cytokines is a defensive process to repair damaged

tissue or whether these cytokines are directly involved

in tissue destruction remains unclear.

The initial success that was seen following the use of

the IL-1 receptor antagonist to reduce infection-medi-

ated in¯ammation, tissue damage and disease severity6

has widened our interest to the speci®c blocking of

many pro-in¯ammatory cytokines.7 Recently, Cominelli

et al. showed that IL-1ra8, 9 and monoclonal antibodies

against IL-810 reduced in¯ammation in immune com-

plex-mediated colitis in rabbits. Since then, researchers

have shown a burgeoning interest in blocking TNFaactivation and binding to its receptors by the use of

various pharmacological approaches. Studies carried

out in natural11 and experimental models of colitis12±16

have provided some evidence of pharmacological suc-

cess with TNFa mab in reducing disease severity. The

success of TNFa mab therapy in such a wide range of

animals attests to the bene®cial effect of this therapy,

irrespective of the mode of induction of colitis. The

mechanism by which TNFa mab induces its therapeutic

effect in these models is not known. However, a recent

study by Pender et al.17 has suggested that TNFa may

cause tissue injury by stimulating the secretion of

matrixmetalloproteinases (MMP), since a p55 TNF

receptor-human gig fusion protein caused a reduction

in MMP release, in vitro. Additionally, open18 and

placebo-controlled trials19, 20 in humans have shown

that the treatment of steroid-resistant Crohn's disease

patients with TNFa mab results in a signi®cant clinical

response and disease remission. These promising results

suggest an escalated future use of TNFa mab in the

treatment of Crohn's disease patients. However, the

mechanism(s) by which these mabs provide their

therapeutic bene®t and the safety of their repeated use

remains to be evaluated.

The short-term blockade of TNFa appears to be

relatively safe. However, there are unpublished anec-

dotal references to these antibodies causing severe side-

effects in a few patients. This makes it unclear whether

a prolonged blockade of TNFa with either a single or

repeated dosing of these long acting TNFa mab will

result in an abnormal host-defence in in¯ammatory

bowel disease. If so, new methods to antagonize the

actions of TNFa that will limit any unwanted side-effects

need to be discovered. Therefore it would be of interest

to ®nd out whether the simultaneous use of TNFarelease inhibitors with TNFa mab will enhance ef®cacy,

reduce the repeated use of these antibodies and abate

the risk of an aberrant complement-mediated host

reactivity. Therefore, the present study was designed

to evaluate the ef®cacy of pentoxifylline, a known TNFarelease inhibitor, when used alone or in combination

with TNFa mab in the dextran sulphate model of

chronic colitis in mice.

MATERIALS AND METHODS

Dextran sulphate (30±40 kDa) was purchased from ICN

Biochemicals, Cost Mesa, CA, USA. Antimouse TNFamab (Lot no. 000034) and ELISA kits for TNFa were

purchased from Endogen, Cambridge, MA, USA. Rat IgG

was purchased from the Sigma Chemical Company (St

Louis, MI).

Animals

Female, 8-week-old, Swiss Webster mice, weighing

25±30 g were used in this study. The animals were

housed in rooms at a controlled temperature of 23 °C,

and light±dark cycles. The mice were fed standard mice

chow pellets, had access to tap water supplied in bottles,

and were acclimated to the vivarium conditions before

they were used in experiments.

Induction of colitis

Mice were fed 3% DSS(3% dextran sulphate solution)

dissolved in drinking water in a cyclic fashion for one to

three cycles, to induce chronic colitis. Each cycle

consisted of giving the mice 3% DSS for 5 days to reach

a disease activity index (DAI) of 1.8±2.0.21 Our prelim-

inary studies showed that at this disease activity level,

the dose of TNFa mab selected was effective in inhibiting

the DAI in DSS-induced mixed in¯ammation. In this

252 S. MURTHY et al.

Ó 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13, 251±260

disease state, all the animals showed loose stools and

were positive for the presence of occult blood in stools;

50% had gross bleeding per rectum. Less than 10% of

the mice had more than 5% weight loss. When the mice

reached the desired DAI, DSS feeding was stopped and

the mice were returned to plain water for 12 days. We21

and others12 have shown that this method of feeding

DSS produces chronic colitis and simulates periods of

quiescence and multiple exacerbations as are observed

in human in¯ammatory bowel disease patients.

Experimental design

The exact experimental design is shown in Figure 1. A

total of seven groups of mice, with each group

consisting of 12 or 24 mice, were tested in this study.

Each group was further divided into six mice per

sacri®ce. In each experimental group, six mice were

sacri®ced at the end of each cycle. The ®rst group of

mice (n � 18) was treated with rat IgG (25 lg/mouse,

0.1 mL, i.p.) at the beginning of each cycle for three

cycles. The second group of mice (n � 18) was treated

with mouse anti-TNFa mab (25 lg/mouse, 0.1 mL, i.p.)

only once at the beginning of the ®rst cycle and not at

all thereafter. The third group of mice (n � 18)

received pentoxifylline (100 mg/kg/day, 0.1 mL, p.o.)

daily, starting at the beginning of the ®rst cycle, and the

therapy was continued until the end of the third cycle.

The fourth group of mice (n � 18) received simulta-

neous treatment with a single injection of anti-TNFamab (25 lg/mouse, 0.1 mL, i.p.) at the beginning of

®rst cycle and daily pentoxy®lline (100 mg/kg/day,

0.1 mL, p.o.) until the end of the third cycle. The ®fth

group of mice (n � 12) received repeated doses of anti-

TNFa mab at the beginning of each cycle for a total of

three repeated doses starting at the beginning of each

cycle. The sixth group (n � 12) received a simulta-

neous treatment of repeated doses of anti-TNFa mab

and a constant dose of pentoxifylline for three cycles.

The seventh group of mice (n � 24) was ®rst exposed

to one cycle of DSS feeding to establish chronic colitis.

At the beginning of second cycle of DSS feeding, the

mice were treated with IgG, TNFa mab or pentoxifylline

alone or TNFa + pentoxifylline to examine the effects of

these mono or combination therapies in mice with

established colitis. The doses of these drugs were the

same as those used before.

Determination of Disease Activity Index (DAI)

Body weights, occult blood or the presence of gross

blood per rectum, and stool consistency were deter-

mined daily. Two investigators (C.M. and C.J.M.) blinded

to the protocol, measured the DAI. The clinical parame-

ters used here are comprehensive functional measures

that are analogous to the subjective clinical symptoms

observed in human ulcerative colitis. This method of

scoring has been validated by repeated studies. It

correlates well with histological healing measured as

crypt scores.21 A signi®cant decrease in the DAI

(P < 0.05) is considered an end-point of successful

therapy.

Histology

At the time of sacri®ce, blood was drawn by cardiac

puncture, and plasma was separated and stored at

)70 °C. After sacri®ce, the colon was removed, from the

colocaecal junction to the anal verge. It was opened

longitudinally along the mesenteric border to examine

gross changes in the appearance of the mucosa. The

colon was divided into three equal pieces to represent

the proximal, middle and distal colon. The colonic

segments were then ®xed separately in buffered 10%

formalin. Processing of colonic tissue is described in

detail in our earlier papers.21, 22 The sections from each

part of the colon were graded for in¯ammation scores

and ulcer index.21, 22

Figure 1. Experimental design, illustrating

treatment groups and sacri®ce times.

DSS� 3% dextran sulphate solution, Ig-

G�Rat IgG 25 lg/mouse, TNFamab�25 lg/mouse, i.p., pentoxifyl-

line�100 mg/kg/day. Groups 1±4: six

animals sacri®ced at the end of each cycle.

*Six mice sacri®ced at the end of the second

and third cycles. ** Daily pentoxifylline

until the end of the third cycle.

ANTI-TNFa COMBINATION THERAPY IN MOUSE COLITIS 253


Determination of plasma TNFa levels

Plasma samples were analysed for circulating TNFalevels using Endogen ELISA kits. Plasma TNFa levels

were measured only in the blood samples of animals

treated with IgG or pentoxifylline.

Statistical analysis

Data are expressed as mean � S.E.M. Statistical analysis

for signi®cant differences was performed using ANOVA

followed by Dunnett's test. Differences were considered

statistically signi®cant when P-values were less than

0.05.

RESULTS

Disease Activity Indexes after single anti-TNFa therapy

The DAI from all groups in each of the DSS cycles is

shown in Figure 2. The DAI on the last day of DSS

feeding reached 1.8±2.0, which is lower than the DAI

traditionally used by us.22, 23

In the ®rst cycle, as shown in Figure 2, the mean DAI

12 days after the ®rst treatment (12 days post-DSS)

with IgG was 1.2 � 0.09. On the same day, mice

treated with TNFa mab alone showed a 42% lower DAI

(P < 0.05) compared to the IgG group. Mice treated

with pentoxifylline alone showed a 44% decrease in DAI

(P < 0.05) compared to IgG group. The combination

therapy of TNFa mab + pentoxifylline inhibited DAI by

57% (P < 0.001). The combination therapy was signif-

icantly better than either TNFa mab or pentoxifylline

alone (P < 0.05).

In the second cycle, 12 days after cessation of DSS, the

mean DAI in animals treated with IgG was 1.6 � 0.02.

On the same day, mice treated with TNFa mab alone,

once at the beginning of the ®rst cycle, showed a 32%

inhibition in DAI, and mice treated with pentoxifylline

alone showed a 30% inhibition in DAI (P < 0.05)

compared to the IgG-treated group. The combination

therapy of TNFa mab + pentoxifylline reduced DAI by

69% (P < 0.001) compared to the IgG-treated group.

The combination therapy was signi®cantly better than

TNFa mab alone (P < 0.05) or pentoxifylline alone

(P < 0.02).

Figure 2. Disease activity indices of mice treated with various anti-TNF and control immunoglobulin (IgG) treatments in each cycle. Each

cycle � 5 days of 3% dextran sulphate feeding + 12 days of water. TNFa mab � 25 lg/mouse, i.p. and pentoxifylline � 100 mg/kg/

day, p.o. throughout the study. *P < 0.05, ** P < 0.001, ***P < 0.0001 signi®cantly lower compared to IgG treated mice. +P < 0.05

signi®cantly higher compared to IgG treated mice. (a) P < 0.05 signi®cantly lower compared to pentoxifylline alone or TNFa mab alone.

(b) P < 0.05 signi®cantly lower compared to second dose of TNFa mab.



In the third cycle, 12 days after stopping DSS dosing,

mice treated with IgG alone had a DAI of 1.2 � 0.18.

On the same day, the single dose of TNFa mab alone

given at the beginning of the ®rst cycle was no longer

effective. Pentoxifylline alone decreased the DAI by 30%

(P < 0.05 compared to IgG) and combination therapy of

TNFa mab + pentoxifylline decreased the DAI by 60%

(P < 0.01 compared to IgG treated group).

DAI after multiple anti-TNFa therapy

In the second cycle, mice treated with a second repeat

dose of TNFa mab alone reduced the DAI by 54%, which

was signi®cantly better (P < 0.05) than a single

administration of TNFa mab and IgG treated groups.

The combination therapy of a repeat second dose of

TNFa mab + pentoxifylline decreased the DAI by 84%.

This decrease was signi®cantly better than a single dose

of TNFa mab alone (P < 0.01), pentoxifylline alone

(P < 0.01) or the single dose of TNFa mab + pentox-

ifylline (P < 0.02).

In contrast, in the third cycle, mice treated with three

repeated doses of TNFa mab given at the beginning of

each cycle worsened the disease, with an increase in

DAI by 30% (P < 0.05 compared to IgG alone) to

1.6 � 0.16. Pentoxifylline alone or a combination

therapy of three repeated dose of TNFa mab + pent-

oxy®lline did not signi®cantly change the DAI compared

to the IgG group.

Anti-TNFa therapy in established disease

In the established model of colitis, TNFa mab alone

reduced the DAI 27%, pentoxifylline alone reduced the

DAI by 20% and the combination therapy reduced the

DAI by 35%, compared to IgG treated group. These

inhibitions were signi®cantly lower (P < 0.05) than

that observed with a single dose of the same agents used

in the ®rst or second cycle.

HistologyÐsingle dose of anti-TNFa therapy

The ulcer index and in¯ammation scores for each

experimental group of mice in all cycles are shown in

Table 1. The mean ulcer index in animals treated with

IgG in the ®rst cycle was 4.3 � 2.6 and the in¯am-

mation score was 16.08 � 0.69. Mice treated with

TNFa mab for one cycle showed 88% and 86.6%

inhibition in ulcer index and in¯ammation score,

respectively. Mice treated with pentoxifylline alone

showed 67.7% and 71.67% inhibition in the ulcer

index and in¯ammation score, respectively. A compar-

ison of the TNFa mab treated group with the

pentoxifylline group did not show any statistical

Table 1. Comprehensive histological scores of mice treated with anti-TNFa therapies

Cycle 1 Cycle 2 Cycle 3

Treatment group UI IS UI IS UI IS

IgG 4.30 � 2.60 16.08 � 0.69 1.80 � 1.06 8.60 � 3.92 3.90 � 1.92 15.11 � 6.60

TNFa mab

(single Injection)

0.50 � 0.34** 2.15 � 3.25** 0.28 � 0.16** 3.14 � 1.32** 0 3.26 � 0.94**

Pentoxifylline (PF)

100 mg/kg/day, p.o.

1.38 � 0.54* 4.56 � 1.41** 0.46 � 0.22** 2.81 � 1.15** 1.46 � 1.09* 8.28 � 3.34*

PF + TNFa mab

(single injection)

0 2.28 � 0.96** 0 3.70 � 1.20** 1.92 � 1.11* 7.54 � 1.11*

TNFa mab

(2 injections)

0.03 � 0.03** 2.95 � 0.93**

TNFa mab

(2 injections) + PF

0.08 � 0.08** 2.50 � 1.28**

TNFa mab

(3 injections)

1.42 � 0.41* 22.18 � 2.70

TNFa mab

(3 injections) + PF

2.00 � 0.36* 14.64 � 3.69

UI � ulcer index; IS � in¯ammation score; IgG � immunoglobulin; TNFa mab � antimouse tumour necrosis factor monoclonal antibody (25 lg/mouse/i.p. injection).

Each cycle � 5 days of 3% dextran sulphate followed by 12 days of water.

*P < 0.05, signi®cantly lower compared to IgG treated group; **P < 0.01, signi®cantly lower compared to IgG treated group and P < 0.05signi®cantly higher compared to IgG group.



difference between groups. Mice treated with pentox-

ifylline + TNFa mab after one cycle showed a complete

absence of ulcerations and a signi®cant inhibition in

the in¯ammation score (85.83%). However, the inhi-

bition in in¯ammation score was not signi®cantly

different compared to TNFa mab alone or pentoxifyl-

line alone, suggesting that the effects we observed were

maximal.

In the second cycle, in the IgG group, mice showed an

ulcer index of 1.8 � 1.06 and an in¯ammation score of

8.6 � 3.92. Mice treated with TNFa mab once during

the ®rst cycle continued to show a signi®cant inhibition

in the ulcer index (84.6%) and in¯ammation score

(63.5%) compared to those animals treated with IgG.

These inhibitions were not signi®cantly different com-

pared to the inhibitions observed at the end of the ®rst

cycle for the same therapies. Mice continuously treated

with pentoxifylline showed a 74.6% inhibition in the

ulcer index and a 67.3% inhibition in the in¯ammation

compared to IgG treated animals. These inhibitions

were not signi®cantly different compared to the inhibi-

tion observed at the end of ®rst cycle for the same

treatment. Mice treated with the combination therapy

of TNFa mab + pentoxifylline showed 100% inhibition

in the ulcer index and 57% inhibition in the in¯amma-

tion score. These inhibitions for ulcer index and

in¯ammation score were similar to those observed in

the ®rst cycle.

In the third cycle, mice treated with a single TNFa mab

injection at the beginning of the ®rst cycle continued to

show a lower ulcer index (60% inhibition) and signi-

®cant inhibition in in¯ammation score (78.14%) com-

pared to the IgG treated group. Mice treated with daily

pentoxifylline alone showed a 62.57% inhibition in

ulcer index, and a 45.2% inhibition in in¯ammation

score. Combination therapy with TNFa mab and

pentoxifylline inhibited the mean ulcer index by

75.67% and in¯ammation score by 80%, which was

signi®cantly lower than TNFa mab alone or pentoxy®l-

line alone. Overall, the histological scores for all groups

were lower than that observed in the ®rst or second

cycle (P < 0.05), suggesting that the effects of these

therapies were either wearing off, or the disease was too

severe at this stage.

HistologyÐmultiple doses of anti-TNFa therapy

In the second cycle, mice treated with a second

repeated dose of TNFa mab showed 98.15% and

65.7% inhibition in ulcer index and in¯ammation

score, respectively, compared to the IgG treated group.

Mice treated with a second dose of TNFa mab +

pentoxifylline showed 95.37% and 70.95% inhibition

in the ulcer index and in¯ammation scores, respective-

ly, compared to IgG treated animals. These inhibitions

were not statistically signi®cant compared to a single

injection of TNFa mab or a single injection of TNFamab + pentoxifylline.

Mice treated with three repeated doses of TNFa mab at

the beginning of each cycle showed an inhibition in

ulcer index of 63.4% compared to the IgG group. The

in¯ammation score was signi®cantly increased by

46.8% compared to the IgG treated group. Similarly,

mice treated with a combination therapy of TNFa mab

and pentoxifylline had an inhibited ulcer index of

48.9%, but this did not inhibit the in¯ammation score

compared to the IgG treated group. Thus, a repeated

injection of TNFa mab caused discrete worsening of

in¯ammation scores.

Qualitative histology

The histological appearance of mice treated with TNFamab and TNFa mab + pentoxifylline in each group and

in each cycle was qualitatively similar. Thus, for

brevity, only the medium power view of a mouse colon

treated with a single dose of TNFa mab at the end of the

third cycle is shown in Figure 3. On the other hand, the

histological appearance of tissues in mice treated with

three repeated doses of TNFa mab, at the end of third

Figure 3. Medium power view of the colon at the end of third cycle

of a mouse treated with a single dose of TNFa mab. The ®gure

shows healing, absence of ulcers, mild in¯ammation and crypt

distortion.



cycle (Figure 4) showed increased ulceration and in-

¯ammation that was worse than that observed with

IgG-treated animals and a single dose of TNFa mab

(Figure 3).

Circulating TNFa levels

In normal non-DSS-fed mice, circulating TNFa levels are

negligible (< 2 pg/mL). Pentoxifylline alone inhibited

circulating TNFa levels, from 210 � 100 pg/mL

(IgG-treated animals) to 33.0 � 39 pg/mL (P < 0.05)

in the ®rst cycle, from 440 � 92 (IgG-treated) to

66 � 55 pg/mL in the second cycle and from

380 � 110 (IgG-treated) to 195 � 70 pg/mL in the

third cycle. Circulating TNFa levels in the TNFa mab

and TNFa + pentoxifylline groups were subject to great

variability, and the values are therefore not presented

here or for other cycles.

DISCUSSION

Recent advances in our understanding of the mecha-

nisms of cytokine release and their control of in¯am-

mation have widened our interest in anticytokine

therapies for the treatment of in¯ammatory bowel

disease. Circulating and local expression or release of

cytokines has been described in in¯ammatory bowel

disease patients24 and in experimental models of

colitis.11, 25 It is now known that these cytokines act

in concert in a complex fashion, inducing pro- and

possibly anti-in¯ammatory effects. Studies carried out

both in experimental animals and in human in¯amma-

tory bowel disease have shown some association

between TNFa and disease.26±29 This association has

been thought to play a key role in promoting in¯am-

mation in the intestines. The clinical success achieved in

Crohn's disease patients with two TNFa mabs in open-

label,18 placebo-controlled trials19, 20 and in experi-

mental models of colitis,12±16 also suggests that TNFamay be a key player in patients with steroid refractory

in¯ammatory bowel disease. Monoclonal antibodies

have demonstrated excellent clinical response and the

maintenance of remission for nearly 12 weeks after a

single injection of TNFa mabs. However, there is a

concern over the use of these antibodies, since they can

produce some adverse side-effects.

The immunological nature and classi®cation of the

antibodies used successfully in Crohn's disease patients

indicate that the cA2 antibodies have both mouse and

human IgG1 templates. Thus, they have a potential to

induce idiotypic antibodies to the mouse region of the

chimera, and antibody dependent cellular cytotoxicity

(ADCC) can be expected. However, there is a speculation

that ADCC may be essential for its therapeutic effect.30

The other antibody, CDP571, is an engineered human-

ized antibody of IgGc1 type comprising of 95% human

and 5% mouse murine sequences and it produces

signi®cantly less or no side-effects.31 Considering the

nature of these antibodies and their relatively longer

half-lives, there is a potential for compromised host

defence in in¯ammatory bowel disease. Thus, a new

approach that facilitates the signi®cant inhibition of

endogenous release of TNFa and the minimal use of

antibodies to neutralize the available TNFa appears

attractive and needs to be tested.

This study makes the ®rst attempt at this combination

therapy using mouse-speci®c TNFa mab with pentoxi-

fylline, in the DSS model of chronic colitis. In this model

the inciting agent was administered repeatedly to

simulate multiple attacks and remissions to produce

the colitis that is reminiscent of human chronic

ulcerative colitis.21

The design of the study includes a single injection of

TNFa mab alone, pentoxifylline alone or a combination

of TNFa mab with pentoxifylline in animals with single

and multiple attacks of colitis. It examines the ef®cacy

and duration of action of a mono or combination

therapy in this model when the therapy is given once or

repeated. The results of this study are consistent with

the observations in humans that a single injection of the

Figure 4. Low power view of the colon at the end of third cycle of

a mouse treated with three doses of TNFa mab (one dose at the

beginning of each cycle). The ®gure shows widespread ulceration,

signi®cant underlying in¯ammation and ulceration.



mouse-speci®c TNFa mab is suf®cient to rapidly sup-

press disease severity and in¯ammation, and to mini-

mize the damage caused by multiple attacks. It also

con®rms the observation made in humans that even the

mouse-speci®c antibodies show a sustained ef®cacy in

this model. Unlike in humans, where the repeated use of

cA2 antibody was tested in remission, we tested the

repeated use of mouse antibody in animals that were

challenged with multiple a exposure to DSS. Despite

this, a single injection of the antibody was quite effective

in inhibiting ulcer index and in¯ammation scores in all

three cycles. We found a slight disparity in the effect of

TNFa mab on DAI vs. the histological scores in the third

cycle after a single injection. Here, while DAI showed no

effect, histological scores were slightly yet signi®cantly

inhibited. We believe that the lack of an effect on the

DAI may be due to incomplete mucosal healing and

in¯ammation in the third cycle that may have contrib-

uted to mucosal permeability and abnormal DAI. Our

studies show that the use of mouse-speci®c mab are not

only effective in inducing remission, but also lessened

the severity of attacks each time these animals were

rechallenged with DSS.

Our studies also show that pentoxifylline is quite

effective in suppressing disease severity, in¯ammation

and ulcer scores in our model. This occurs in association

with a decrease in circulating TNFa levels. This

observation is consistent with previous studies which

showed that pentoxifylline is an effective inhibitor of

TNFa release. Interestingly, pentoxifylline in combina-

tion with a single injection TNFa mab is signi®cantly

more effective in inhibiting the disease severity, ulcer

index and in¯ammation in all three cycles compared to

a placebo or any single monotherapies. This result is of

interest, since the combination therapy enhanced the

ef®cacy in all three cycles and reduced the adverse pro-

in¯ammatory effect of a repeated use TNFa mab in the

third cycle.

A recent study carried out using the biopsy samples

from in¯ammatory bowel disease patients showed that

pentoxifylline is an effective inhibitor of TNFa release,

in vitro.32 However, pentoxifylline at 400 mg/kg, given

four times a day, failed to reduce disease severity in

active Crohn's disease patients and did not change the

dose of steroids.33 The investigators in this study33 did

not examine stimulated TNFa release from the mono-

cytes isolated from these patients. Therefore, it is

dif®cult to determine whether pentoxifylline actually

inhibited the release of TNFa from the local mono-

cytes.34 Be that as it may, this ®nding dampens the

hypothesis of whether the inhibition of TNFa release is

an effective therapeutic method for achieving a good

clinical response in Crohn's disease. Circulating TNFalevels have not been great predictors of disease severity

or therapeutic success.26±29 On the contrary, local

release and paracrine effects of TNFa has been suggested

in experimental models and in¯ammatory bowel disease

patients. Thus, agents that inhibit the local release of

TNFa may make TNFa mab more ef®cient and enhance

its therapeutic ef®cacy.

The observation that, in spite of prolonged ef®cacy, the

need for a repeated use of these antibodies to maintain

remission, requires a careful evaluation of adverse

effects before such therapy can be implemented. Inter-

estingly, in two of the studies in which a repeated use of

the antibody was tested,35, 36 the antibody was effective

in prolonging remission but not disease severity. In fact,

the results of our study demonstrated that a repeated

use of this antibody in the third cycle evoked an

unwanted in¯ammatory reaction. Since the antibody

we have used in this study is mouse-speci®c, these

results should be interpreted with caution, when they

are extended to other antibodies made speci®cally for

human use. Since the ef®cacy or the adverse effects of

chimeric or engineered TNFa mab has only been tested

using remission protocols, it is dif®cult to make any

judgement on the repeated use of TNFa mab at the time

of an acute or multiple attacks.

Previous studies have shown that the release of TNFafrom monocytes is regulated by the enzyme phospho-

diesterase IV37 that prevents the breakdown of cAMP

leading to elevated intracellular cAMP levels. Pentoxi-

fylline, a xanthine derivative, is also a good inhibitor of

PDEIV, but it is less speci®c than some of the more

speci®c inhibitors such as rolipram.38 Whether the low

speci®city of pentoxifylline had anything to do with the

overall lack of response in Crohn's disease patients

remains unknown. The disparity between our study and

the study in Crohn's disease patients33 is obvious, since

in our study, pentoxifylline alone inhibited disease

severity and improved histological scores. It is possible

since the DSS model of colitis has several resemblances

to ulcerative colitis, whether this proclivity towards

ulcerative colitis had any in¯uence on the outcome

needs to be further investigated in this model and in

human ulcerative colitis. In human ulcerative colitis,

TNFa mab have provided only partial success, and

speci®c TNFa release inhibitors have never been tested.



The observations made in this study with the combina-

tion therapy are provocative and suggest the testing of

other forms of anti-TNFa therapies in humans to

enhance, prolong and limit any side-effects associated

with TNFa therapy.

ACKNOWLEDGEMENT

This study was supported by Krancer Center for IBD

Research.

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Combination therapy of pentoxifylline and TNFα monoclonal antibody in dextran sulphate-induced mouse colitis