European Journal of Surgical Oncology xxx (xxxx) xxx

Contents lists available at ScienceDirect

European Journal of Surgical Oncology

journal homepage: www.ejso.com

A novel treatment of bromelain and acetylcysteine (BromAc) in

patients with peritoneal mucinous tumours: A phase I first in man
study
S.J. Valle a, J. Akhter a, A.H. Mekkawy a, S. Lodh b, K. Pillai a, S. Badar a, D. Glenn b,
M. Power b, W. Liauw c, D.L. Morris a, d, *
a
Department of Surgery, St George Hospital, Kogarah, NSW, Australia
b
Department of Radiology, St George Hospital, Kogarah, NSW, Australia
c
Cancer Care Centre, St George Hospital, Kogarah, NSW, Australia
d
Department of Surgery, University of New South Wales St George Clinical School, Kogarah, NSW, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Background: Bromelain (Brom) and Acetylcysteine (Ac) have synergistic activity resulting in dissolution
Received 11 September 2019 of tumour-produced mucin both in vitro and in vivo. The aim of this study was to determine whether
Accepted 25 October 2019 treatment of mucinous peritoneal tumour with BromAc can be performed with an acceptable safety
Available online xxx
profile and to conduct a preliminary assessment of efficacy in a clinical setting.
Methods: Under radiological guidance, a drain was inserted into the tumour mass or intraperitoneally.
Keywords:
Each patient could have more than one tumour site treated. Brom 20e60 mg and Ac 1$5-2 g was
Mucinous peritoneal tumour
administered in 5% glucose. At 24 h, the patient was assessed for symptoms including treatment-related
Mucolytic drug
Bromelain
adverse events (AEs) and the drain was aspirated. The volume of tumour removed was measured. A
Acetylcysteine repeat dose via the drain was given in most patients. All patients that received at least one dose of
Cytoreductive surgery and intraperitoneal BromAc were included in the safety and response analysis.
chemotherapy Findings: Between March 2018 and July 2019, 20 patients with mucinous tumours were treated with
BromAc. Seventeen (85%) of patients had at least one treatment-emergent AE. The most frequent
treatment-related AEs were CRP rise (n ¼ 16, 80%), WCC rise (n ¼ 11, 55%), fever (n ¼ 7, 35%, grade I) and
pain (n ¼ 6, 30%, grade II/III). Serious treatment-related AEs accounted for 12$5% of all AEs. There were no
anaphylactic reactions. There were no deaths due to treatment-related AEs. An objective response to
treatment was seen in 73$2% of treated sites.
Conclusion: Based on these preliminary results and our preclinical data, injection of BromAc into
mucinous tumours had a manageable safety profile. Considerable mucolytic activity was seen by volume
of mucin extracted and radiological appearance. These results support further investigation of BromAC
for patients with inoperable mucinous tumours and may provide a new and minimally invasive treat-
ment for these patients.
© 2019 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical
Oncology. All rights reserved.

Introduction
Pseudomyxoma peritonei (PMP) is a rare syndrome involving
secretion of mucin by tumours, which progressively fills the
abdominal cavity. PMP tumour most commonly arises from
* Corresponding author. Department of Surgery, Level 3 Pitney Building, St
George Hospital, Gray Street, Kogarah, NSW, 2217, Australia.
E-mail address: David.Morris@unsw.edu.au (D.L. Morris).
tumours of the appendix but can rarely originate from other sites
including ovary, bowel and urachus [1].
The management of peritoneal spread of mucinous tumours,
including pseudomyxoma peritonei (PMP) and some other tumours
that spread to the peritoneum, has been transformed by the Sug-
arbaker procedure consisting of complete cytoreductive surgery
and intraperitoneal chemotherapy (CRS/IPC). This major procedure
has shown to benefit many patients with a median survival
extending beyond 16 years [1].

https://doi.org/10.1016/j.ejso.2019.10.033
0748-7983/© 2019 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
2 S.J. Valle et al. / European Journal of Surgical Oncology xxx (xxxx) xxx
Unfortunately, almost half of patients that undergo this pro-
cedure with appendix tumours recur [2,3]. Mucinous tumours may
produce widely disseminated mucinous deposits that mimic ascites
or isolated intra-abdominal collections of mucin, of varying cellu-
larity. Unlike ascites, these deposits are not able to be easily drained
or aspirated due to the high viscosity created by the mucin. Repeat
surgeries for recurrence are of increasing surgical complexity,
increased morbidity, prolonged hospitalisation and a progressively
smaller chance of cure [4]. Patients may benefit from palliative
surgery, however most will only be deemed suitable for 2e3 such
procedures before becoming inoperable [5]. Appendix tumours
causing PMP are classified into low and high grade, however even
recurrent low-grade mucinous tumours are progressively debili-
tating and ultimately fatal due mainly to compression, sepsis and
malnourishment. There is currently no pharmacological treatment
for recurrent PMP with mucin itself being a cause of resistance to
cytotoxics [6].
We have previously described remarkable synergy between two
agents, Bromelain (Brom), a mixture of enzymes derived from the
pineapple stem, and Acetylcysteine (Ac), which affected tumour
produced mucin in vitro and in vivo [7e10]. Later, we found that
BromAc had a cytotoxic and chemosensitising effect on solid
gastrointestinal tumours by removing MUC1, MUC2 and MUC5AC,
mucins associated with invasiveness, metastasis, cellular prolifer-
ation and chemoresistance of gastrointestinal tumours [11e13]. We
have shown that BromAc inhibits in vitro and in vivo growth of
human gastrointestinal cancers [9] and is also synergistic with
certain chemotherapy agents [9e14].
The projected benefits of applying BromAc in patients with
inoperable mucinous tumours include the potential to improve the
progressive debilitating symptoms of these diseases such as pain,
vomiting, inability to maintain an adequate oral intake, and pro-
gressive loss of general condition that eventuates in mortality. We
describe the first clinical results of a direct or intraperitoneal in-
jection of BromAc for treatment of inoperable mucinous tumours.
Methods
All patients were seen by a dedicated multidisciplinary team
(MDT) experienced in the management of peritoneal disease.
Eligible patients considered for BromAc treatment had inoperable
mucinous tumour or declined surgery, were at least 18 years old,
had no unmanageable coagulation disorder and did not have an
allergy to the Ananas comosus group, eggs or sulphur.
Patients had baseline blood tests including full blood count, liver
and renal function, c-reactive protein (CRP), fibrinogen and coag-
ulation screen. A computed tomography (CT) scan was performed
and reviewed by an MDT to assess tumour burden, accessibility and
the dimensions and volume of tumour were calculated. Patients
underwent education about the procedure by the principal in-
vestigators and nurse prior to consent.
On the day of the drain insertion procedure, the patient was
fasted for 6 h prior and premedicated with oral antihistamine
(Loratidine 10 mg). Under CT guidance, one or more drains were
placed directly into the cystic tumour mass (directly into the spe-
cific targeted collection of mucinous disease) or free intraperito-
neally by one of three experienced interventional radiologists (IR).
Following placement, the IR attempted aspiration of the drain to
ensure tumour could not be aspirated prior to drug treatment as a
baseline measurement. In some cases, contrast was injected prior
to drug to ensure optimal distribution. BromAc was then admin-
istered by the IR.
The doses of Brom 30e45 mg (intratumoural) or 45e60 mg
(intraperitoneal) (Mucpharm Pty Ltd, Australia) and Ac 1$5 g
(intratumoural) or 1$5-2 g (intraperitoneal) (Acetylcysteine-Link,
Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
Link Pharma, Australia) were reconstituted and administered in 5%
glucose through a sterile Millex 0$22mm syringe filter to remove
Brom debris. The volume injected, where possible, was equal to 20%
of the target tumour volume (intratumoural) or 500 ml (intraper-
itoneal). Drug doses were determined based on estimated tumour
volume (Brom 30 mg NAC 1$5 g tumour <250 ml, Brom 45 mg NAC
1$5 g tumour >250 ml or multiple treatment sites, intraperitoneal
Brom 45 mg NAC 1$5 g tumour burden low or Brom 60 mg NAC 2 g
extensive tumour. In patients that had more than one drain placed,
the drug was administered between the sites based on the volume
of tumour at each. The maximum daily dose of Brom and Ac were
60 mg and 2 g, respectively. The drain was capped following drug
injection and remained in situ for access.
All patients were monitored post procedure with routine clinical
observations including blood pressure, heart and respiratory rates,
temperature, oxygen saturation, pain score and blood test. The
patient was assessed for treatment related symptoms or side ef-
fects. A blood sample was separated and stored for measurement of
drug levels. The patient was discharged from outpatient care or
admitted to the ward for observation if there were any clinical
concerns.
The following day (24 h post procedure), the patient was
assessed for symptoms or treatment related side effects. The drain
site was assessed and aspirated. The volume of tumour removed
was measured. Repeat treatment at 24 h was then considered.
Repeat treatments were administered via the drain, as described
above. Samples of aspirated fluidised tumour were collected for
laboratory analysis of drug concentrations and where appropriate,
microbiology.
All treatment related symptoms or side effects were reported to
the sponsor and serious adverse events to the Human Research and
Ethics Committee and graded using the Common Terminology
Criteria for Adverse Events (CTCAE) [15]. RECIST v1$1 criteria were
utilised where possible to measure objective response and pro-
gression free survival, as previously described [16]. In patients that
did not meet RECIST v1$1 criteria, aspiration volume following drug
treatment was utilised as a marker of response (aspiration of >25%
of calculated baseline tumour volume) and stable disease was
defined as no change on CT appearance or no greater than 20%
increase in sum of dimensions and absolute increase of 5 mm and/
or volume during follow up. Follow up time was measured from the
date of the last drug injection. Repeat blood tests were performed
at 1 week and at 1 month with a CT scan. Residual tumour volume
was calculated and monitored by follow up 3 monthly CT scan and
blood test, or as clinically indicated. Patients were asked to com-
plete quality of life questionnaires including the RAND measure of
health-related quality of life short form survey (SF-36), report on
their symptoms and a treatment survey. Ethics approval for this
study was provided by Bellberry Ltd (2017-07-561) and patients
that did not meet criteria including retreatments were conducted
via compassionate access using the same protocol and reporting
requirements. All procedures were conducted within ethical stan-
dards. This trial is registered at ClincialTrials.gov (NCT03976973)
and anzctr.org.au (ACTRN12617001612303).
Results
Twenty-three patients were consented for treatment. Two pa-
tients were unsuitable for treatment at drain insertion with sus-
pected gastrointestinal fistulation of tumour evident upon injection
of contrast and one patient pursued alternate non-pharmacological
therapies. Overall, 20 patients underwent 28 treatments with
BromAc from March 2018 to July 2019. Thirteen (65%) patients had
intratumoural treatment and 7 patients (35%) had intraperitoneal
treatment. The primary tumour was low grade PMP (n ¼ 6),
 Table 1
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous

Patient treatment characteristics, schedule and outcomes following treatment with BrNAC.

Patient # Tumour Treatment # of treat Location Repeat Cumulative dose Volume Response Status post treatment
sites ments treatment aspirated
Br (mg) NAC (g)
(#. of drains) (ml)

1 AppCa 4 8 IP No 270 10.5 4465 Partial response, symptomatic improvement Died day 144 (refused further
73% reduction, hard disease omentum, no response. Stable treatment)
disease at 3 months
2 AppCa 1 3 IT No 90 3.0 262 Partial response, symptomatic improvement Alive with disease day 513
pelvis 47% reduction (Fig. 1b)
Stable disease at 15 months, on follow up
3 AppCa Hard 2 8 IP No 220 13.0 958 Partial response gastric, symptomatic improvement, 42% Died day 212 (lack of nutritional
tumour reduction (Figs. 1a and 3a,b) no response omentum support)
Stable disease at 3 months, progressive disease at 4 months
4 PMP 1 2 IT No 60 3.0 17 No response, drain dislodged, unable to aspirate. On follow up, Alive with disease day 445
pelvis stable disease at 15 months
5 PMP 2 2 IT Yes 60 3.0 52 Partial response, symptomatic improvement Alive with disease day 301
Hard tumour 2 pelvis Day 192 60 3.0 185 46% reduction

S.J. Valle et al. / European Journal of Surgical Oncology xxx (xxxx) xxx
Progressive disease at 6 months, retreated day 192, partial
response 52% reduction
Progressive disease at 6 months, on follow up
6 PMP 1 4 IT No 120 4.0 57 Near complete response, symptomatic improvement, >90% Free of macroscopic disease e had
liver hilus reduction surgery day 305
Progressive disease at 9 months
7 PMP 5 3 IT Yes 90 3.0 110 Near complete response, >95% reduction, symptomatic Alive with disease day 291
7 abdo wall, Day 262 210 10.5 742 improvement (Fig. 1c)
pelvis Progressive disease at 6 months and new sites of disease. Repeat
treatment day 262, partial response 48e71% reduction, on
follow up
8 PMP 4 2 IT Yes 60 2.0 350 Partial response, 76% reduction symptomatic resolution Free of macroscopic disease e had
Hard tumour 2 Pelvis, liver Day 112 183 60 3.0 145 Progressive disease at 3 months, retreated day 112, partial surgery day 352
6 hilus 259 180 9.0 280 response 62% reduction, symptomatic improvement
6 165 7.5 775 Progressive disease at 2 months, retreated day 183, partial
response 71% reduction, symptomatic improvement
Progressive disease at 2 months, retreated day 259, partial
response 86% reduction, symptomatic resolution, no response
hilus (Fig. 3d)
9 OvCa 1 4 IP No 210 7.5 17610 Near complete response apart from ovarian primary tumour Died day 166 (refused treatment for
(not targeted), symptom resolution (Fig. 2) urinary sepsis)
10 CRC 1 1 IT No 30 1.5 0 No response, intent of treatment to relieve compression of Underwent stoma revision day 4
stoma stoma, tumour found to be invading serosa
11 OvCa 3 2 IT Yes 60 3.0 660 Partial response, symptomatic improvement Alive with disease day 248
2 peri gastric, Day 219 60 3.0 412 91% reduction Non-compliant
pelvis Stable disease 6 months with new disease elsewhere e repeat
treatment new sites day 219, on follow up
12 PMP 1 2 IP No 60 3.0 2220 Response based on aspiration volume e free intraperitoneal, Died day 97, palliated (not disease
symptomatic improvement related)
13 AppCa 2 2 IT Yes 60 3.0 24 Partial response, 34% reduction Alive with disease day 224
Hard tumour 2 liver hilus Day 175 60 3.0 85 Progressive disease 3 months retreated day 175, ceased
treatment e allergic reaction, no response
14 AppCa 1 2 IT No 60 3.0 65 Partial response, 61% reduction Alive with disease day 119
Hard tumour right abdo Ceased treatment, fistula from tumour erosion
15 OvCa 2 9 IP No 450 15.0 23705 Response based on aspiration volume, free intraperitoneal, Died day 53, aspiration pneumonia
symptomatic improvement
(continued on next page)

3
 4 S.J. Valle et al. / European Journal of Surgical Oncology xxx (xxxx) xxx

appendix adenocarcinoma (n ¼ 10), mucinous ovarian tumours

(n ¼ 3) and colon cancer (n ¼ 1). The mean number of operations

Alive with disease day 73 e had

Alive with disease day 62 e had

prior to treatment was 3$8 (range 2e9).
Adverse events occurred in 85% of patients (n ¼ 17). Overall,
Alive with disease day 238

Alive with disease day 126

Alive with disease, day 13

there were 88 adverse events of which 11 were serious treatment-
Status post treatment

related adverse events (12$5%). Fevers were experienced by 35%

(n ¼ 7), grade II/III pain at the drain site or during injection was

surgery day 52

surgery day 19
described in 30% (n ¼ 6) of patients. Hospital admission occurred in

Key: AppCa e appendix adenocarcinoma, PMP e pseudomyxoma peritonei, OvCa e ovarian cancer, CRC e colorectal cancer, IT e intratumoural, IP e intraperitoneal, abdo e abdominal, # - number.
3 patients (15%) as precautionary measure following treatment. A
liver haematoma (n ¼ 1, transhepatic drain placement), hypo-
volemia (n ¼ 1) and peritoneal inflammation (n ¼ 1) were seen. A
gastrointestinal fistula (n ¼ 1) (managed by existing drain until
Partial response based on aspiration volume in liver, pelvis. No
Partial response perigastric, 49% reduction complete response

closure) and bladder hole (n ¼ 1) (managed by insertion of an

No response, extensive hard disease. Unable to aspirate any
Debulking surgery day 52 for bowel obstruction, hard mass
Partial response based on aspiration volume, symptomatic

indwelling catheter) occurred as a consequence of removing

tumour from a pre-existing site of tumour invasion (Table 2). All
3 month scan new sites of disease, treated day 151

adverse events were manageable.

response mass above bladder. Awaiting follow up

An objective response to BromAc treatment was seen in 73.2% of

treated sites (30 of 41 sites (85% of patients)) following a mean of
Partial response, symptomatic resolution

Partial response, symptomatic resolution

4$2 doses (mean 3.1 intratumoural, 5.8 intraperitoneal). Similarly,

abdo wall (Fig. 3c), awaiting follow up

the length of treatment varied dependent on tumour burden, with

3 month scan new site of disease

Table 2
Description and occurrence of adverse events following treatment with BrNAC.
fluid. Palliative surgery.

Patients receiving Grade

BrNAC therapy
around small bowel

(n ¼ 20)
59% reduction

39% reduction

improvement.

Number of patients with adverse events 17 (85%)

All AEs
Response

C-reactive protein increase 16 (80%) e

White cell count increase 11 (55%) e
Fever 7 (35%) I
Pain 6 (30%) II/III
aspirated
Volume

Liver enzyme derangement (increase) 6 (30%) I

3743

2875

Platelet count increase 6 (30%) I

(ml)

967
76
93

Pain 5 (25%) I
0

Nausea 4 (20%) I
NAC (g)
Cumulative dose

Hypoalbuminemia 4 (20%) II
Vomiting 3 (15%) I
4.5
4.5

6.0

6.0

6.0

7.5

Gram negative tumour cavity infection 2 (10%) II

Dehydration (from nausea/vomiting) 1 (5%) III
Br (mg)

Abdominal distension 1 (5%) II

120

210

180

180

210
80

Wound drain site infection 1 (5%) II

Sepsis (abdominal) 1 (5%) III
Pleural effusion (allergic reaction*) 1 (5%) I
treatment

Operating theatre washout of infected 1 (5%) II

Day 151
Repeat

tumour cavity
Yes

No

No

No

No

Presyncope 1 (5%) II
Syncope 1 (5%) III
above bladder &

Allergic reaction (inflammation)* 1 (5%) I

IT peri-gastric,

Anaemia 1 (5%) II
liver, pelvis,

Hypovolemia 1 (5%) II
abdo wall
Location

Bladder hole (from tumour erosion) 1 (5%) II

pelvis

Gastrointestinal fistula (from tumour erosion) 1 (5%) II

IP

IP

IP
IT

IT

Liver haematoma 1 (5%) I

Chills 1 (5%) II
# of treat

Back pain 1 (5%) I

ments

Skin irritation (drain site dressing) 1 (5%) I

Flushing 1 (5%) I
3
5

AE leading to discontinuation
(#. of drains)

Allergic reaction 1 (5%) I

Treatment

Gastrointestinal fistula (tumour erosion) 1 (5%) II

Serious AEs
sites

Albumin infusion (hypoalbuminemia) 2 (10%) II

4

Sepsis (abdominal) 1 (5%) III

Hard tumour

Hard tumour

Operating theatre washout of infected 1 (5%) II

tumour cavity
Tumour

Bladder hole 1 (5%) II

AppCa

AppCa

AppCa

AppCa

AppCa
Table 1 (continued )

Gastrointestinal fistula 1 (5%) II

Dehydration requiring admission for IV fluids 1 (5%) III
Anaemia requiring transfusion 1 (5%) II
Patient #

Hypovolemia (extensive tumour removal) 1 (5%) II

Wound drain site infection 1 (5%) II
16

17

18

19

20

Liver haematoma 1 (5%) I

Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
 S.J. Valle et al. / European Journal of Surgical Oncology xxx (xxxx) xxx 5

a mean treatment time of 3$4 (range 3e7 days) and 11$5 days
(range 9e24 days) for intratumoural and intraperitoneal targets,
respectively. The total tumour aspirates varied from 0 to 23,705 ml
(Table 1, Figs. 1e3). The viscosity of the aspirated tumour varied
from quite thick gelatinous material after initial treatment to pro-
gressively more fluid with additional treatments. There were
technical failures at two sites with drain dislodgement and three
sites were had hard tumour without response. Two sites had Bro-
mAc injection only without drain placement. Surgery occurred in 6
patients post treatment 30% (delayed debulking surgery n ¼ 3,
bowel obstruction n ¼ 1, stoma revision n ¼ 1, washout for sepsis
n ¼ 1).
A rise in CRP was seen in almost all patients (n ¼ 16, 80%) which
ranged from 78$1e7102$4% from baseline. Either or both white cell
count (33$4e114$8%) and neutrophils (22$7e109$3%) were also
elevated in 11 of these patients (69%). Similarly, aspartate amino-
transferase (AST), alanine aminotransferase (ALT), gamma gluta-
myltransferase (GGT) and alkaline phosphatase (ALP) and platelets
were transiently increased above laboratory range in 30% of pa-
tients (n ¼ 6) above laboratory range. Hypoalbuminemia was seen
in 20% of patients (n ¼ 4), all of whom had extensive peritoneal
tumour removal. There were otherwise no significant changes in
coagulation or blood profile. Further, there were no drug-related
deaths due to treatment-related adverse events.
Fifteen patients (75%) reported a reduction in tumour-related
symptoms at the time of discharge compared to pre-treatment.
Fourteen (70%) patients completed quality of life forms at base-
line, 1 month and 3 months post procedure. There was no delete-
rious effect on quality of life.
Blood samples and aspirated tumour were collected for assay
with sensitivity to Brom 50 ng/ml, we detected Brom in serum
blood taken 3, 6, and 12 h following treatment ranging from
53$2e128 ng/ml. There was no Brom detected in 24 h samples. In
the tumour aspirated from the drain (24 h after treatment), Brom
was detected in 39 aspirates taken 24 h post treatment ranging
from 0 to 202mg/ml.
Discussion
This is the first attempt at percutaneous dissolution of highly
mucinous tumours by means of percutaneous intratumoural or
intraperitoneal injection of BromAc. Our drug treatment is inten-
ded to dissolve mucinous tumour masses, allowing for extraction.
Brom is a combination of enzymes from the pineapple (Ananas
comosus) stem, which hydrolyses glycosidic bonds in mucin, whilst
Ac reduces disulphide bonds in mucin, resulting in dissolution of
mucinous mass. Additionally, this combination of mucin-targeting
drug treatment also affects the tumour's key biological structure
and protective framework (oncoproteins), resulting in considerable
cytotoxicity [9].
Brom is a widely used drug, generally regarded as safe and is
mainly taken orally as a digestive aid. It has also been approved for
topical use in 3rd degree burns where up to 51 g of Brom has been
applied topically in one treatment, achieved with safety (EMA
648483/2012) [17]. Prior to this, the safety of Brom by intravenous
injection in mini pigs and rodents was assessed. The major concern
with parenteral administration of Brom has been its effects on
coagulation (due to its fibrinolytic activity). Brom has very low oral
toxicity with an LD50 (lethal doses) greater than 10 g/kg in mice,
rats, and rabbits [17e20]. Dosages of 1500 mg/kg per day when
administered to rats showed no carcinogenic or teratogenic effects
and did not provoke any alteration in food intake, histology of
heart, growth, spleen, kidney, or haematological parameters.
Similarly, Ac is widely used for paracetamol/acetaminophen
overdose by intravenous injection, where 300 mg/kg is given in the
first 24 h [21] and in a 70 kg patient, 10$5 g would be administered.
Up to 18% of patients experience anaphylactic reaction to Ac. Lower
incidence has been reported with slower rates of infusion [22].
In the current single centre study, adult patients with PMP or
other mucinous tumours who were not candidates for CRS/IPC or
other debulking surgery, had a soft to intermediate viscosity mucin,
and do not meet any of the exclusion criteria, received treatment
with BromAc. We report the relative safety of this approach and an

Fig. 1. a) Aspiration of fluidised mucin 24 h following injection of BrNAC; b) pre- (top) and post-treatment (bottom) scan performed at 512 days of a male patient with an appendix
adenocarcinoma pelvic recurrence; and c) pre- (top) and immediately post-treatment scan (drain in situ) of a female patient with an abdominal wall appendix tumour recurrence
compressing her stoma site.

Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
6 S.J. Valle et al. / European Journal of Surgical Oncology xxx (xxxx) xxx

Fig. 2. Pre- (left) and 1-month (right) post-treatment (non-contrast) scan of a patient with ovarian cancer that underwent intraperitoneal BrNAC treatment (x2) for thick mucinous
ascites.

Figure 3. pre- (a) and post-treatment scan performed at 1 month (b) of a male patient with a symptomatic appendix adenocarcinoma recurrence compressing his stomach; c)
aspiration of lightly blood stained tumour in a patient with appendix tumour recurrence 24 h following injection of BrNAC; and d) day 2 aspirates (24 h following injection of
BrNAC) in a male patient with recurrent appendix adenocarcinoma in the pelvis.

objective response rate in a significant proportion of patients.
The current maximum dose per treatment in our study is Brom
60 mg and Ac 2.0 g, well within these known safety limits although
the safety of a combination of BromAc or intraperitoneal admin-
istration in patients was unknown. The side effects seen in our
study have been manageable. A transient but sharp CRP rise is an
effect of treatment, thought to be due to the dissolution of the
tumour and inflammation at the treatment site or cavity. The white
cell count, neutrophil, platelet and liver function derangement are
expected to be a response to the CRP and inflammatory changes.

Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
 S.J. Valle et al. / European Journal of Surgical Oncology xxx (xxxx) xxx
Similarly, small amounts of blood-stained fluid removed may also
be a result of this phenomenon. Interestingly, both Brom and Ac are
described as having anti-inflammatory properties [23,24]. In all
patients, blood parameters returned to baseline on their 1-month
follow up blood test.
The possibility of post procedural intraabdominal sepsis or
wound infection due to pre-existing bowel wall erosion of tumour
and subsequent removal of tumour acting as a barrier between the
bowel and peritoneum will require monitoring. Similarly, a
communication of the tumour treated with the urinary bladder was
seen in one patient, who was managed with an indwelling catheter
until resolved. Caution should be taken in cases where there is
suspected fistulous connections, especially tumour invasion of the
gastrointestinal tract as a fistula may be created when the tumour is
dissolved. The risks of treatment with BromAc also include hyper-
sensitivity to either drug. One patient in this study reported an
intolerance to eggs, which have a high content of thiol. Upon
administration of the drug, flushing was reported with generalised
pain that may have been evoked by the thiol (acetylcysteine). A CT
scan revealed peritoneal inflammation.
Injury to bowel or other structures during the interventional
procedure is possible, as with any percutaneous abdominal pro-
cedure, and this should only be performed by experienced IRs. This
treatment requires a close and collaborative multidisciplinary team
in deciding whether a patient is truly inoperable, the trajectory and
location of the drain placement and post procedural care with the
competency to manage potential complications. The role of IR in
oncology already includes biopsy, fluid drainage, percutaneous
tumour thermal ablation, selective arterial drug delivery, and may
possibly be extended to percutaneous “debulking” with BromAc.
Limitations of BromAc direct tumour injection or peritoneal
application include the difficulty of differentiation between the
various grades of hardness in mucin by radiological imaging. It is
clear from our in vitro and in vivo studies that the drug combination
is much more effective in the soft textured mucinous tumours,
which are present in up to 70% of cases [7,8,25,26]. Softer tumours
are considerably easier to dissolve than hard tumours, which may
be related to known biochemical differences, the increased cellu-
larity seen in harder tumours or ability for drug to disseminate
easier. There may however be a role for repeated treatments in
patients with hard texture or compact tumours or combining
BromAc as a sensitiser with chemotherapy. We have reported sig-
nificant inhibition in a range of cancer types by BromAc both in vitro
and in vivo and have observed profound increase in sensitivity to a
range of chemotherapy agents [14,27]. Another limitation is that
some patients have intratumoural/intramucinous fibrous locula-
tion, potentially making treatment and aspiration more difficult.
These patients present with complicated disease and differing
recurrence patterns following multiple abdominal interventions,
therefore treatment plan, safety and efficacy from this procedure
may vary. Patients with thin disease, such as disease lining the
small bowel without a tumour cavity cannot be treated via direct
injection due to the inability to place a drain in the tumour. The role
of intraperitoneal delivery in these patients is being assessed and
was safely achieved in our three patients with large volumes of
liquified tumour aspirated. We are now expanding our clinical
studies to examine the role of BromAc in combination with
chemotherapy in patients with inoperable peritoneal metastases
and the potential of incorporating this regimen into an earlier
phase treatment, such as prior to or during CRS/IPC.
The intent of this paper is not to report on long term outcomes;
however, it is remarkable that a number of patients remain alive
and clinically well for more than 1 year after treatment. Ultimately,
the rationale for injection of BromAc directly into tumour re-
currences or intraperitoneally is to dissolve the tumour-produced
Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
7
mucin and allow for its extraction as a relatively minimally inva-
sive treatment to relieve compressive effects. Whilst the objective
response rate in this study was high, this may not translate into a
major clinical advantage in patients with extensive disease. We
acknowledge that this is a relatively small study, which patients are
most likely to benefit from this treatment and for how long is
currently unknown.
Conclusion
This is the first study on the dissolution of highly mucinous
tumours by percutaneous intratumoural or intraperitoneal injec-
tion of BromAc. Our previous investigation on the mucolytic effect
of BromAc due to the synchronous breakage of glycosidic linkages
and disulphide bonds supported the use of this treatment in pa-
tients with inoperable mucinous tumours. We saw no anaphylaxis
or serious reactions; however this is certainly possible with either
of the drugs. At present, little is known about optimal dosage, safety
in treating certain areas of the peritoneum and repeat treatments.
However, from these preliminary results, BromAc treatment of
inoperable mucinous tumours had a manageable safety profile with
an objective response in a substantial number of patients based on
mucin extracted and radiological appearance. Similarly, whilst the
long-term clinical benefit is unknown, these results support further
investigation of BromAc for inoperable mucinous tumours and may
provide a new and minimally invasive treatment option for these
patients.
Funding
This work was supported by Mucpharm Pty Ltd; The David
Morris Cancer Research Fund (UNSW Foundation/Division of Phi-
lanthropy); and the Appendix Cancer and Pseudomyxoma Peritonei
(ACPMP) Research Foundation.
Declaration of competing interest
Professor D.L. Morris is the co-inventor and assignee of the in-
tellectual property and co-owner and director of the sponsor
company, Mucpharm Pty Ltd. Ms S.J. Valle is co-owner of Muc-
pharm Pty Ltd. Dr J. Akhter, Dr K. Pillai are employed by Mucpharm
Pty Ltd and co-inventors of the intellectual property. Professor D.L.
Morris and the study team guarantees that the conduct of this
study is in accordance with protocol, ethics and national and gov-
ernment (hospital) guidelines and regulations.
Acknowledgements
The David Morris Cancer Research Foundation (administered
through the University of New South Wales, Division of Philan-
thropy) through which patients and their families have supported
our laboratory research for many years. The Appendix Cancer and
Pseudomyxoma Peritonei (ACPMP) Research Foundation and Na-
tional Organization for Rare Disorders (NORD) for research grant
(16-002). Sponsor Mucpharm Pty Ltd. The St George Hospital
Department of Surgery Peritonectomy Unit and Radiology Depart-
ment. Dr Glenn & Partners Kogarah NSW Australia for use of radi-
ology facilities. The University of New South Wales (NSi) has
assigned the IP through license 2017-0045 to Professor D.L. Morris
(inventor).
References
[1] Chua TC, Moran BJ, Sugarbaker PH, Levine EA, Glehen O, Gilly FN, et al. Early-
and long-term outcome data of patients with pseudomyxoma peritonei from
8 S.J. Valle et al. / European Journal of Surgical Oncology xxx (xxxx) xxx
appendiceal origin treated by a strategy of cytoreductive surgery and hyper-
thermic intraperitoneal chemotherapy. J Clin Oncol 2012;30(20):2449e56.
[2] Smeenk HG, van Eijck CH, Hop WC, Erdmann J, Trab KC, Debois M. Long-term
survival and metastatic pattern of pancreatic and periampullary cancer after
adjuvant chemoradiation or observation: long-term results of EORTC trial
40891. Annals of Surgery 2007;246(5):734e40.
[3] Miner TJ, Shia J, Jaques DP, Klimstra DS, Brennan MF, Coit DG. Long-term
survival following treatment of pseudomyxoma peritonei: an analysis of
surgical therapy. Annals of Surgery 2005;241(2):300e8.
[4] Chua TC, Quinn LE, Zhao J, Morris DL. Iterative cytoreductive surgery and
hyperthermic intraperitoneal chemotehrapy for recurrent peritoneal metas-
tases. J Surg Oncol 2013;108(2):81e8.
[5] Jarvinen P, Ristima €ki A, Kantonen J, Aronen M, Huuhtanen R, J€arvinen H, et al.
Comparison of serial debulking and cytoreductive surgery with hyperthermic
intraperitoneal chemotherapy in pseudomyxoma peritonei of appendiceal
origin. Int J Colorectal Dis 2014;29(8):999e1007.
[6] Jonckheere N, Skrypek N, van Seuningen I. Mucins and tumour resistance to
chemotherapeutic drugs. Biochim Biophys Acta Rev Canc 2014;1846(1):
142e51.
[7] Pillai K, Akhter J, Chua TC, Morris DL. A formulation for in situ lysis of mucin
secreted in pseudomyxoma peritonei. Int J Cancer 2014;134(2):478e86.
[8] Pillai K, Akhter J, Chua TC, Morris DL. Potential mucolytic agents for mucinous
ascites from pseudomyxoma peritonei. Investig New Drugs 2012;30(5):
2080e6.
[9] Amini A, Masoumi-Moghaddam S, Morris DL. Utility of bromelain and N-
acetylcysteine in treatment of peritoneal dissemination of gastrointestinal
mucin-producing malignancies. London: Springer; 2016.
[10] Pillai K, Pourgholami MH, Chua TC, Morris DL. MUC1 as a potential target in
anticancer therapies. Am J Clin Oncol: Cancer Clin Trials 2015;38:108e18.
[11] Amini A, Masoumi-Moghaddam S, Ehteda A, Liauw W, Morris DL. Depletion of
mucin in mucin-producing human gastrointestinal carcinoma: results from
in vitro and in vivo studies with bromelain and N-acetylcysteine. Oncotarget
2015;20(6):33329e44.
[12] Amini A, Masoumi-Moghaddam S, Morris DL. In vivo assessment of growth-
inhibitory and mucin-depleting effects of bromelain and n-acetylcysteine in
peritoneal carcinomatosis models. Ann Oncol 2015;26(2). ii28.
[13] Amini A, Masoumi-Moghaddam S, Ehteda A, Morris DL. Bromelain and N-
acetylcysteine inhibit proliferation and survival of gastrointestinal cells
in vitro: significance of combination therapy 2014;33(1):92.
[14] Amini A, Ehteda A, Masoumi Moghaddam S, Akhter J, Pillai K, Morris DL.
Cytotoxic effects of bromelain in human gastrointestinal carcinoma cell lines
(MKN45, KATO-III, HT29-5F12, and HT29-5M21. OncoTargets Ther
2013;16(6):403e9.
Please cite this article as: Valle SJ et al., A novel treatment of bromelain and acetylcysteine (BromAc) in patients with peritoneal mucinous
tumours: A phase I first in man study, European Journal of Surgical Oncology, https://doi.org/10.1016/j.ejso.2019.10.033
[15] https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/
CTCAE_v5_Quick_Reference_8.5x11.pdf. [Accessed 3 May 2019].
[16] Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al.
New response evaluation criteria in solid tumours: revised RECIST guideline
(version 1.1). Eur J Cancer 2009;45:228e47.
[17] EMA/6488483/2012 NexoBrid: concentrate of proteolytic enzymes enriched
in bromelain, public assessment report. European Medicines Agency; 20
September 2012. . [Accessed 13 April 2018].
[18] Moss JN, Frazier CV, Martin GM. Bromelains. The pharmacology of the en-
zymes. Arch Int Pharmacodyn Ther 1963;145:166e89.
[19] Bromelain Taussig SJ. A proteolytic enzyme and its clinical application. A re-
view. Hiroshima J Med Sci 1975;24(2):185e93.
[20] Castell JV, Friedrich G, Kuhn CS, Poppe GE, et al. Intestinal absorption of
undegraded proteins in men: presence of bromelain in plasma after oral
intake. Am J Physiol 1997;273(1):G139e46.
[21] Daly F, Fountain J, Murray L, Graudins A, Buckley NA, Panel of Australian and
New Zealand clinical toxicologists. Guidelines for the management of para-
cetamol poisoning in Australia and New Zealand e explanation and elabo-
ration. Med J Aust 2008;188(5):296e302.
[22] Bateman N, Dear JW, Thanacoody KH, Thomas SH, Eddleston M,
Sandilands EA, et al. Reduction of adverse effects from intravenous ace-
tylcysteine treatment for paracetamol poisoning: a randomised controlled
trial. The Lancet 2014;383(9918):697e704.
[23] Taussig SJ, Batkin S. Bromelain, the enzyme complex of pineapple (Ananas
comosus) and its clinical application: an update. J Ethnopharmacol
1988;22(2):191e203.
[24] Palacio JR, Markert UR, Martinez P. Anti-inflammatory properties of n-ace-
tylcysteine on lipopolysaccharide-activated macrophages. Inflamm Res
2011;60(7):695e704.
[25] Huang Y, Alzahrani NA, Fisher OM, Chua TC, Kozman MA, Liauw W, et al.
Intraoperative macroscopic tumour consistency is associated with overall
survival after cytoreductive surgery and intraperitoneal chemotherapy for
appendiceal adenocarcinoma with peritoneal metastases: a retrospective
observational study. Am J Surg 2019;217(4):704e12.
[26] Karpes J, Parikh R, Shamavonian R, Alzahrani NA, Morris DL, et al. Analysis of
feasibility and survival in repeated cytoreductive surgery and hyperthermic
intraperitoneal chemotherapy (CRS/HIPEC) in appendiceal cancer with peri-
toneal dissemination. J Clin Orthod 2019:e15702.
[27] Amini A, Masoumi-Moghaddam S, Ehteda A, Liauw W, Morris DL. Potentiation
of chemotherapeutics by bromelain and n-acetylcysteine: sequential and
combination therapy of gastrointestinal cancer cells. American Journal of
Cancer Research 2016;6(2):350e69.