Histamine reduces BNCT induced mucositis in precancerous tissue

without affecting BPA biodistribution or long term inhibition of tumor development

A. Monti Hughes1, E.C.C. Pozzi1, S. Thorp1, P. Curotto1, V.A. Medina2,3, D.J. Martinel Lamas2, E.S. Rivera2, M.A. Garabalino1, E.M. Heber1, M.E. Itoiz1,4,

R.F. Aromando1,4, D.W. Nigg5, V.A. Trivillin1,3, A.E. Schwint1,3.

1National Atomic Energy Commission (CNEA), Argentina; 2School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), Argentina; 3National Research Council (CONICET), Argentina; 4Faculty of Dentistry, UBA, Argentina, 5Idaho National Laboratory (INL) USA.

Squamous cell carcinoma of the head and neck (HNSCC) remains a major cause of morbidity and mortality worldwide.

The relatively poor overall 5-year survival rate for malignancies of the oral cavity poses the need for more effective and selective therapies.

To date, BNCT clinical results have shown a potential therapeutic advantage, with room for improvement.

Studies in appropriate experimental models are pivotal to progress in this field.

HEAD AND NECK CANCER

SYRIAN GOLDEN HAMSTER CHEEK POUCH ORAL

CANCER MODEL

Mesocricetus auratus

Non-cancerized hamster cheek pouch (everted).Mesocricetus auratus

SYRIAN GOLDEN HAMSTER CHEEK POUCH ORAL CANCER MODEL Widely known animal system that

closely mimics events involved in the development of premalignant and malignant human oral lesions.

Similar to human oral mucosa (in histological, histochemical and ultrastructural terms).

Resembles a pocket of oral mucosa in the thickness of the hamster´s cheek.

It does not develop spontaneous tumors.

It can be easily everted for local irradiation and follow-up.

Applied without anesthesia.

Cancerized hamster cheek pouch with a tumor (arrow) surrounded by precancerous tissue.

Unlike models of implanted tumor cells in normal tissue, it mimics the spontaneous process of malignant transformation.

Provides a tumor model surrounded by precancerous tissue which gives rise to the formation of additional tumors, as occurs in field cancerized human oral mucosa in

head and neck cancer.

SYRIAN GOLDEN HAMSTER CHEEK POUCH ORAL CANCER MODELCARCINOGENESIS PROTOCOL: Repeated topical application of the carcinogen

dimethyl-1,2-benzanthracene (DMBA)

Topical application of DMBA 0.5% in mineral oil.

The study of precancerous tissue in our hamster model is useful because:

The development of tumors from precancerous tissue would model the development of second primary tumors and recurrences in field cancerized tissue in human oral

mucosa in head and neck cancer, a frequent cause of therapeutic failure.

Toxicity in precancerous tissue mimics the constraints imposed on therapeutic protocols by the dose limiting

nature of field cancerized tissue in humans.

Short-term (1 month) therapeutic efficacy of BPA-BNCT, GB-10-BNCT and (GB-10+BPA)-BNCT to treat hamster cheek pouch oral cancer with no normal tissue radiotoxicity, and slight/moderate mucositis in dose-limiting precancerous tissue around tumors (Kreimann et al, 2001a; Trivillin et al, 2006; Pozzi et al., 2009).

Previous studies

Classical carcinogenesis protocol (12 weeks, twice a week) was useful for short-term tumor control studies (1 month follow up) but:

• Does not allow long-term studies of precancerous tissue, which are essential to model (with constraints) a clinical scenario in which the risk of developing second primary tumors jeopardizes therapeutic efficacy;• Does not mimic the kinetics of human oral carcinogenesis.

Oral precancer model (6 weeks, twice a week) for long-term follow up (8 months) (Heber et al., 2010)

Long-term therapeutic efficacy of Double (GB-10+BPA)-BNCT 10 Gy total absorbed dose to precancerous tissue (in two 5 Gy doses, 4 weeks apart). (Monti Hughes et al., 2013)

In a clinical scenario, oral mucositis limits the dose delivered to head and neck tumors and affects patients’

quality of life.

Despite therapeutic success, BNCT-induced mucositis in precancerous tissue was dose limiting and favored, in some

cases, tumor development (Monti Hughes et al., 2013).

Despite many years of research there are few radiation protectors in use, whose clinical value is limited due to

their toxicity.

Nowadays mucositis continues to represent an important unmet medical need in oncology practice.

The aim of the present study was to evaluate the effect of the administration of radioprotective

agents, seeking to reduce the incidence of severe BNCT-induced mucositis in precancerous tissue.

RADIOPROTECTIVE AGENTS:HISTAMINE

JNJ7777120JNJ10191584

Protect small intestine, bone marrow and salivary gland from gamma radiation in mice and rats, with no local or systemic side effects.

(Medina et al., 2007, 2011; Martinel Lamas et al., 2013)

DMBA-cancerized animals (Oral precancer carcinogenesis protocol: 6 weeks, twice a week) treated with BPA-BNCT 5 Gy total absorbed dose AND:

RADIOBIOLOGICAL STUDIES

Group 1) HISTAMINE LOW CONCENTRATION (1mg/kg, sc, n=6 animals)

Group 2) HISTAMINE HIGH CONCENTRATION (5mg/kg, sc, n=6 animals)

CONTROL GROUP: vehicle (saline solution) (n=11 animals)

Group 3) JNJ7777120 (10mg/kg, sc, n=7 animals)

Group 4) JNJ10191584 (10mg/kg, sc, n=3 animals)

Cancerized, sham irradiated animals (no BNCT or radioprotective treatment) (n=88 animals)

All radioprotectors were applied 1 day before BNCT, on the day of BNCT (concomitantly with BPA injection) and daily for 14 days after BNCT.

BPA-BNCT protocol: the incidence of severe mucositis in field cancerized tissue was highest with this protocol in our previous studies (Monti Hughes et al., 2013).

Thermal column Shutter External shield

water

core

Irradiation position

Sample insertion

RA-3 (Buenos Aires, Argentina)

Thermal column

Sample insertion

Irradiation position

Sample insertion

The animals were irradiated at the RA-3 thermal facility.

The animals are inserted into a near-isotropic neutron field while the reactor is in normal operation.

RADIOBIOLOGICAL STUDIES – RA-3 thermal facility

The animals were placed inside a Lithium-6 carbonate shielding to protect their bodies while the cheek pouch is everted out of the enclosure onto a protruding shelf for exposure.

RADIOBIOLOGICAL STUDIES – Animal shielding

RADIOBIOLOGICAL STUDIES

Absorbed Doses (Gy) for each of the Dose Components:Fast

neutronsGamma photons

Boron (precancerous tissue)(per ppm) Induced protons

BPA-BNCT (15.5 mg10B/kg b.w.)Cancerized

cheek pouch

- 0.36±0.09 0.30±0.07 0.9±0.1

Boron concentration in

precancerous tissue

Irradiation time

Thermal neutron fluxat the irradiation position

Mean absorbed

dose

12.5±2.6 ppm 7.9 min (7.7±0.6)*109 n.cm-2.seg-1 5.0±1.7 Gy

Dose calculations were performed using the “Hamster Tool” software developed by Farías R and Gonzalez S.

Must be minimally toxic itself.

IDEAL RADIOPROTECTOR

Must reduce the incidence of severe mucositis.

Must not compromise BNCT therapeutic effect.

FOLLOW UP 8 months after BNCT

Grade 0: healthy

appearance, no

erosion or vasodilatio

n.

Grade 1: erythema

and/or edema and/or

vasodilation, no

evidence of mucosal erosion.

Grade 2: severe

erythema and/or edema,

vasodilation and/or

superficial erosion.

Grade 3: severe

erythema and/or edema,

vasodilation and

formation of ulcers <2

mm in diameter.

Grade 4: severe

erythema and/or edema,

vasodilation and

formation of ulcers ≥2 mm and <4

in diameter,

and/or necrosis

areas <4mm in

diameter.

Grade 5: severe

erythema and/or edema,

vasodilation and

formation of ulcers ≥4mm or multiple

ulcers ≥2mm, and/or

necrosis areas

≥4mm in diameter. Grading was based on the most severe feature.

Radiotoxicity in terms of mucositis in precancerous tissue: six grade oral mucositis scale based on macroscopic features adapted from other studies in humans and non-cancerized hamsters (Lopez-Castaño et al., 2005; Sonis et al., 2000):

Inhibitory effect of BNCT on the development of new tumors in precancerous tissue.

Local and systemic side effects.

SEVERE MUCOSITIS: G4/G5

% of animals affected by severe mucositis (G4/G5) and % of animals with new tumors after irradiation (8 months follow up).

RESULTS For all radioprotective agents: only local signs of irritation.

SEVERE MUCOSITIS(Grade 4/Grade 5)

% ANIMALS WITH NEW TUMORS (8

months after BNCT)

CONTROL GROUP: BPA-BNCT + saline 55% 67%

BPA-BNCT + Histamine LOW concentration 17% 40%

BPA-BNCT + JNJ10191584 100% 50%

BPA-BNCT + JNJ7777120 57% 50%

BPA-BNCT + Histamine HIGH concentration 67% 50%

Cancerized, sham irrad (no treatments) 0% 89%

HisLOW was able to reduce the incidence of severe BNCT induced mucositis vs Control group. Both JNJ treatments and HisHIGH were not able to reduce it.

None of the protocols compromised the therapeutic effect of BNCT in terms of inhibition of tumor development from precancerous tissue at 8 months after treatment.

Hamster cheek pouch treated with BPA-BNCT without radioprotective treatment (saline sol.) vs. BPA-BNCT + HisLOW at 14 days post treatment (time at which maximum mucositis was observed).

BPA-BNCT 5Gy + HisLOW

GRADE 2BPA-BNCT 5Gy + saline solution

GRADE 5

Necrosis

RESULTS

Focal erosion

TissueBPA + saline solution* BPA + Hislow**

Mean±SD (ppm) Mean±SD (ppm)

Tumor 10±3 (n=3) 17±6 (n=5)

Precancerous tissue 16±10 (n=2) 12±3 (n=4)

Normal pouch tissue 10±3 (n=2) 14±2 (n=4)

Blood 6±1 (n=2) 6±2 (n=4)

“n” is number of samples; * 2 animals; **4 animals.

BIODISTRIBUTION STUDIES

Treatment with Hislow did not significantly alter gross boron concentration from BPA in tumor, precancerous tissue, normal pouch tissue or blood (p≥0.11).

The use of histamine LOW concentration reduced the incidence of BNCT-induced severe mucositis associated with BPA-BNCT at 5 Gy total dose without compromising the inhibitory effect of BNCT on tumor development. This could be due to:

> The role of Histamine as a radical oxygen species (ROS) scavenger.> The capacity of histamine to act as a growth factor, favoring

proliferation and repair and inhibiting apoptosis.

DISCUSSION

Biodistribution studies showed that Hislow did not affect gross boron biodistribution in tumor or field cancerized tissue. This is an asset in terms of preserving therapeutic efficacy.

Long periods of high histamine concentration (5 mg/kg) and JNJ compounds (10mg/kg) failed to exert a radioprotective effect. This could be due to:

> A dose dependent effect on tumor development. An increase in proliferation rate could be enhancing radiosensitivity and consequently an increase in severe mucositis.

> Extensive removal of ROS would affect the healing process of mucositis.

Two assets to consider histamine as a radioprotector in BNCT of head and neck tumors:

Medina et al. (2011b) have shown that histamine also prevents functional and morphological radioinduced alterations of submandibular glands.

A synthetic derivative of histamine, histamine dihydrochloride, is currently safely used in clinical trials (Romero et al., 2009).

DISCUSSION

This study suggests the potential use of treatment with

histamine LOW concentration (1mg/kg) to minimize the

incidence of severe mucositis in field cancerized tissue

associated with BPA-BNCT at a therapeutic absorbed

dose of 5 Gy, in an oral precancer model.

These results may be of clinical value in reducing

radiation radiotoxicity in head and neck cancer patients

undergoing BNCT treatment.

CONCLUSION

THANK YOU!Contact info: andre.mh@gmail.com

andrea.monti@cnea.gov.ar

Los Alerces National Park; Patagonia ARGENTINA

ACKNOWLEDGMENTS

Medina et al. (2011b) have shown that histamine also prevents functional and morphological alterations of submandibular glands induced by ionizing radiation and completely reversed the radiation-induced reduced salivation in rats.

Overall, histamine would not only reduce oral mucositis in BNCT of head and neck cancer but could also be used to preserve submandibular salivary glands

and prevent xerostomia in patients with salivary gland carcinoma and head and neck cancers.

A synthetic derivative of histamine, histamine dihydrochloride, is currently safely used in clinical trials as an adjuvant for the potential treatment of different cancers, exhibiting no unexpected or irreversible side effects (Romero et al. 2009).

This would be an asset in considering the use of histamine as a radioprotector in BNCT of head and neck tumors.

DISCUSSION

JNJ7777120

JNJ10191584

Human oral mucosa Hamster oral mucosa

Thin non-keratinized or parakeratinized layer. Epithelium witdh: 200 mm.Below: Connective tissue, muscule and the skin of the cheek. Approximately: 2000 mm of thickness.

Orthokeratinized layer. Epithelium witdh: 30 - 50 mm.Below: Connective tissue, interrupted muscule and a loose adventitia. Approximately: 300 - 600 mm of thickness.