Protective Effect of a Mitochondria-Targeted Peptide against the

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The protective effect of a mitochondria-targeted peptide against the development of chemotherapy-induced peripheral neuropathy in mice Satoshi Toyama, Naohito Shimoyama, H. H. Szeto, Peter W. Schiller, and Megumi Shimoyama ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00013 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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ACS Chemical Neuroscience

1

Title

2

The protective effect of a mitochondria-targeted peptide against the development of

3

chemotherapy-induced peripheral neuropathy in mice

4 5

Authors

6

Satoshi Toyama,a,b Naohito Shimoyama,c Hazel H. Szeto,d Peter W. Schiller,e,f Megumi

7

Shimoyamac*

8 9

a

Minato-Ku, Tokyo 105-8471, Japan

10 11

b

c

Department of Palliative Medicine, Jikei University Hospital, 3-19-18 Nishi-Shimbashi, Minato-Ku, Tokyo 105-8471, Japan

14 15

Department of Critical Care and Anesthesia, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-Ku, Tokyo 157-8535, Japan

12 13

Department of Neuroscience, Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi,

d

Burke Medical Research Institute, 785 Mamaroneck Avenue

16

White Plains, NY 10605, USA

17

e

Laboratory of Chemical Biology and Peptide Research, Montreal Clinical Research

18

Institute, 110 Pine Avenue West, Montreal, Que., Canada H2W 1R7

19

f

20

H3C 3J7, Canada

Department of Pharmacology and Physiology, University of Montreal, Montreal, Quebec,

21 22 23 24

* Corresponding author

25

Megumi Shimoyama, MD, PhD

26

Department of Palliative Medicine

27

Jikei University Hospital

28

3-19-18 Nishi-Shimbashi, Minato-Ku, Tokyo 105-8471, Japan

29

Tel: +81-3-3433-1111

30

Fax: +81-3-5400-1247

31

E-mail address: [email protected]

32

ACS Paragon Plus Environment

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1

Abstract

2

Several chemotherapeutic agents used for cancer treatment induce dose-limiting peripheral

3

neuropathy that compromises patients’ quality of life and limits cancer treatment. Recently,

4

mitochondrial dysfunction has been shown to be involved in the mechanism of

5

chemotherapy-induced peripheral neuropathy. SS-20 is a mitochondria-targeted peptide

6

that promotes mitochondrial respiration and restores mitochondrial bioenergetics. In the

7

present study, we examined the protective effect of SS-20 against the development of

8

chemotherapy-induced peripheral neuropathy utilizing a murine model of peripheral

9

neuropathy induced by oxaliplatin, a first-line chemotherapy agent for colon cancer. Weekly

10

administrations of oxaliplatin induced peripheral neuropathy as demonstrated by the

11

development of neuropathic pain and loss of intraepidermal nerve fibers in the hind paw.

12

Continuous

13

oxaliplatin-induced neuropathic pain and mitigated the loss of intraepidermal nerve fibers to

14

normal levels. Our findings suggest that SS-20 may be a drug candidate for the prevention

15

of chemotherapy-induced peripheral neuropathy.

administration

of

SS-20

protected

against

the

development

of

16

17

Keywords

18

Oxaliplatin, chemotherapy, chemotherapy-induced peripheral neuropathy, pain, neuropathic

19

pain, mitochondria

20


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1

Chemotherapy

agents

such

as

paclitaxel,

oxaliplatin,

and

vincristine,

cause

2

dose-dependent peripheral neuropathy i.e., chemotherapy-induced peripheral neuropathy

3

(CIPN) that compromises patients’ quality of life during cancer treatment and may lead to

4

changes in treatment to lower doses or non-neurotoxic agents with obvious negative

5

implications for disease outcomes. Pain due to CIPN is often difficult to treat, and duloxetine,

6

a serotonin-norepinephrine reuptake inhibitor, has been shown to have some effect in

7

relieving pain due to CIPN.1 It is the sole treatment that has been recommended by the

8

American Society of Clinical Oncology (ASCO) for the treatment of CIPN with a moderate

9

strength of recommendation.2 On the other hand, the ASCO guideline states that there is no

10

established agent recommended for the prevention of CIPN. Thus, the development of

11

innovative strategies to prevent CIPN is warranted.

12

In order to study the mechanism and develop treatment of CIPN, we developed a murine

13

model of CIPN using oxaliplatin, a first-line chemotherapy agent against colon cancer.3 This

14

model was carefully designed to closely mimic clinical conditions. The mice gradually

15

developed neuropathic pain which was assessed by the presentation of hypersensitivity of

16

the hind paw to mechanical stimuli, a typical symptom observed in patients. Morphological

17

studies showed that while there were no overt changes in the DRG neurons and peripheral

18

nerves innervating the hind paw, there was loss of intraepidermal nerve fibers (IENFs) of the



1

skin, which has been reported in other animal models of CIPN and in patients with CIPN.4,5

2

Although the mechanism of CIPN is still unclear, it has been shown that mitochondrial

3

dysfunction caused by mitotoxic effects of cancer chemotherapeutic agents is an important

4

component of the dysregulation in primary afferent sensory neurons.3,4,6,7 Studies with

5

animal models suggest that several drugs that prevent mitochondrial injury or improve

6

mitochondrial function may be effective in the treatment of CIPN.3,4,6 In our animal model of

7

oxaliplatin-induced neuropathic pain,3 a tetrapeptide, SS-31 (Figure 1a), which targets and

8

concentrates on the inner mitochondrial membrane, the site of the electron transport chain

9

and reactive oxygen species (ROS) production,8 was shown to protect against the

10

development of neuropathic pain. In addition, SS-31 prevented the loss of IENFs in our

11

study. IENFs are likely to have high energy consumption due to frequent turnover of the skin

12

and to be vulnerable to energy deficiency. SS-31 is a ROS scavenger and has also been

13

shown to improve mitochondrial bioenergetics. Thus, it is unclear whether the protective

14

effect of SS-31 against the development of CIPN was due to the ability to promote

15

mitochondrial respiration or the ability to scavenge ROS. Another tetrapeptide, SS-20

16

(Figure 1b), is an analogue of SS-31, which has the same property of promoting

17

mitochondrial bioenergetics but does not have ROS scavenging capacity.9 In the present

18

study we examined the protective effect of SS-20 against the development of CIPN using


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1

our murine model in order to determine whether oxaliplatin-induced peripheral neuropathy

2

involves mitochondrial bioenergetics deficit and to examine whether SS-20 may be a drug

3

candidate for the prevention of CIPN.

4

5

RESULTS AND DISCUSSION

6

CIPN was induced in mice by 3 weekly administrations of oxaliplatin at 10 mg/kg and was

7

assessed behaviorally by the development of neuropathic pain and morphologically by the

8

loss of IENFs. The protective effect of SS-20 was examined by continuously administering

9

the compound subcutaneously throughout the course of CIPN development and comparing

10

the degree of CIPN developed with that in mice that received continuous administration of

11

vehicle. Neuropathic pain was manifested by hypersensitivity of the paws to non-noxious

12

mechanical and cold stimuli. The mechanical hypersensitivity was assessed by the

13

decrease in mechanical threshold to withdraw the hind paw when touched by von Frey

14

filaments and the cold hypersensitivity was assessed by the increased pain-related behavior

15

(shaking of the forepaw) in response to 15℃ stimulation, a temperature that does not

16

normally cause pain. Mice were administered SS-20 (5 mg/kg/day or 10 mg/kg/day) or

17

saline via subcutaneously implanted Alzet pumps. Three weekly administrations of

18

oxaliplatin decreased paw withdrawal mechanical thresholds compared to baseline in all



1

mice, demonstrating the development of mechanical hypersensitivity (0.02±0.01 vs.

2

0.90±0.21 g, p = 0.001 for vehicle group; 0.30±0.16 vs. 1.12±0.42 g, p < 0.001 for mice

3

given SS-20 at 5 mg/kg/day; 0.64±0.40 vs. 1.06±0.61 g, p = 0.032 for mice given SS-20 at

4

10 mg/kg/day) (Figure 2a). However, the paw withdrawal mechanical thresholds after

5

oxaliplatin administrations were significantly higher in mice that were administered SS-20 at

6

10 mg/kg/day compared to the vehicle group (0.64±0.40 vs. 0.02±0.01 g, p = 0.009) (Figure

7

2a). In the mice that were administered vehicle continuously, 3 weekly administrations of

8

oxaliplatin significantly increased the number of forepaw shakes induced by 15 ℃

9

stimulation as compared to baseline, demonstrating the development of cold

10

hypersensitivity (post-oxaliplatin vs. baseline: 25±9 vs. 9±4 /2.5 min, p < 0.001) (Figure 2b).

11

The continuous administration of SS-20 inhibited the increase in forepaw shakes (SS-20 at

12

5 mg/kg/day vs. vehicle, 14±7 vs. 25±9 /2.5 min, p = 0.002; SS-20 at 10 mg/kg/day vs.

13

vehicle, 12±5 vs. 25±9 /2.5 min, p < 0.001) (Figure 2b). In mice that were continuously

14

administered SS-20 at 10 mg/kg/day, the number of forepaw shakes after 3 weekly

15

administrations of oxaliplatin was not different from baseline values (post-oxaliplatin vs.

16

baseline: 12±5 vs. 8±1 /2.5 min, p = 0.058) (Figure 2b). These results indicate that

17

continuous SS-20 treatment dose-dependently attenuates the development of mechanical

18

and cold hypersensitivity induced by repeated oxaliplatin administrations.


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1

The effect of continuous SS-20 treatment on the changes in IENF density induced by

2

three weekly administrations of oxaliplatin were examined (Figure 3 and 4). In mice that

3

were administered vehicle, the density of IENF markedly decreased compared to naïve

4

mice 7 days after the last injection of oxaliplatin (2.7±1.5 vs. 13.3±2.6 fibers/mm, p = 0.003).

5

The decrease in IENF density was markedly mitigated in mice that were continuously

6

administered SS-20 at 10 mg/kg/day and IENF density in these mice was not different from

7

that in naïve mice (SS-20 treatment vs. vehicle treatment: 12.7±3.2 vs. 2.7±1.5 fibers/mm, p

8

= 0.006; mice with SS-20 treatment vs. naïve mice: 12.7±3.2 vs. 13.3±2.6 fibers/mm, p =

9

1.000).

10

The effect of acute administration of SS-20 on established oxaliplatin-induced neuropathic

11

pain was examined. The acute administration of SS-20 at 10 mg/kg did not affect

12

mechanical (Figure 5a) and cold hypersensitivity (Figure 5b) that had been induced by 3

13

weekly administrations of oxaliplatin (SS-20 treatment vs. vehicle treatment: 0.06±0.06 vs.

14

0.04±0.05 g, p = 0.917 for paw withdrawal mechanical threshold; SS-20 treatment vs.

15

vehicle treatment, 21±6 vs. 23±5 /2.5 min, p = 0.559 for paw shakes induced by 15℃

16

stimulation).

17

These results demonstrated that continuous administration of SS-20 prevented the

18

development of neuropathic pain and the loss of IENFs induced by repeated administration



1

of oxaliplatin, but SS-20 did not affect neuropathic pain symptoms after they were

2

established. Thus it is indicated that SS-20 is capable of preventing oxaliplatin-induced

3

peripheral neuropathy.

4

Recently, mitochondrial dysfunction and oxidative stress have been shown to be involved

5

in neuropathic pain10,11 and in chemotherapy-induced neuropathies.3,4,6,7 Chemotherapeutic

6

agents, such as oxaliplatin and paclitaxel, cause functional impairment of peripheral nerve

7

mitochondria, with significant decrease in state 3 respiration and adenosine triphosphate

8

(ATP) production,4,7 and the mitochondrial dysfunction leads to electron leak and increased

9

production of ROS.4,6,7 The resultant bioenergetic deficiency likely causes abnormal

10

spontaneous discharge in the axons of somatosensory primary neurons due to ion-pumping

11

deficiency.12 The specific mechanism by which oxaliplatin and paclitaxel inhibit

12

mitochondrial bioenergetics and promote ROS production is not known, but cisplatin is

13

known to interact with cardiolipin present in the inner mitochondrial membrane, resulting in

14

cardiolipin peroxidation, loss of cardiolipin, respiratory inhibition, glutathione depletion, and

15

apoptosis.7 Cardiolipin plays important roles in cristae formation as well as in the

16

organization and function of respiratory supercomplexes.

17

SS (Szeto-Schiller) peptides are cell-permeable tetrapeptides that target mitochondrial

18

cardiolipin. SS-20 has been shown to bind to and interact with cardiolipin and modulate


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1

cytochrome c/cardiolipin complex activity resulting in promotion of oxidative phosphorylation,

2

reduced ROS production, and prevention of the conversion of cytochrome c from an

3

electron carrier to a peroxidase, thereby preventing cardiolipin peroxidation.13 By protecting

4

cardiolipin, SS-20 protects cristae membranes and improves mitochondrial respiration and

5

oxidative phosphorylation coupling during ischemia.14 Unlike SS-31, SS-20 cannot

6

scavenge electrons, but it is still a very effective mitochondria-targeted antioxidant because

7

it can reduce ROS production and prevent cytochrome c peroxidase activity. SS-20 is as

8

effective

9

1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine in mice.15 In particular, both SS-20 and SS-31

10

prevented mitochondrial swelling and inhibition of mitochondrial respiration and ATP

11

production induced by 1-methyl-4-phenylpyridium ion in isolated mitochondria and neuronal

12

cell cultures.

as

SS-31

in

protecting

striatal

dopamine

neurons

against

13

The central nervous system has also been shown to be involved in oxaliplatin-induced

14

neuropathy. Glial activation in the spinal cord and different areas of the brain was observed

15

in rats given repeated administration of oxaliplatin.16 Furthermore, N-palmitoylethanolamine,

16

an endogenous amide, was shown to protect against glial activation in the central nervous

17

system as well as against the development of neuropathic pain in rats that were

18

administered oxaliplatin repeatedly.17 In an in vitro study, oxaliplatin was suggested to cause



1

damage to mitochondria of astrocytes,18 which may be associated with astrocyte activation

2

induced by oxaliplatin in vivo. SS-20 crosses the blood brain barrier, and has been shown to

3

have protective effects in a murine model of Parkinson’s disease which involve

4

mitochondrial dysfuction.15 Thus the effects of SS-20 on neuropathic pain symptoms

5

observed in the present study may also involve its protective effects on astrocyte

6

mitochondria in the central nervous system.

7

The present findings with SS-20 are similar to our earlier report that prophylactic

8

administration of SS-31 can attenuate the pain due to oxaliplatin-induced neuropathy and

9

mitigate the loss of IENFs.3 It should be noted that information on the effect of SS-20 on

10

tumor growth is lacking and would be important for SS-20 to have therapeutic use. SS-31,

11

on the other hand, had no effect on tumor growth in mice with and without oxaliplatin

12

treatment (personal communication, Fabio Penna, University of Turin, Italy). Future studies

13

are needed to address this point. Nonetheless, our results suggest that mitochondrial

14

bioenergetics deficit is involved in the mechanism of CIPN and that SS-20 may be a

15

promising drug candidate for the prevention of CIPN.

16

17

CONCLUSIONS


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1

In a murine model of oxaliplatin-induced peripheral neuropathy, continuous administration

2

of SS-20 protected against the development of oxaliplatin-induced neuropathic pain and

3

mitigated the loss of IENFs. Our findings suggest that promotion of mitochondrial respiration

4

is a high-potential strategy to prevent CIPN, and SS-20 may be a promising drug candidate

5

for its prevention.

6

7

METHODS

8

Animals and drugs. Male BALB/c mice, 8 weeks old at the time of first oxaliplatin

9

administration, were used in the experiments. All mice were housed on a 12:12 h dark–light

10

cycle with food and water ad libitum. Experiments were approved by the Institutional Animal

11

Use Committee of Jikei University (Tokyo, Japan), and conducted in accordance with the

12

National Institutes of Health guidelines and the International Association for the Study of

13

Pain Committee for Research and Ethical Issues guidelines for animal research.19

14

Oxaliplatin was obtained from Yakult Co., Ltd. (Tokyo, Japan) and was dissolved in distilled

15

water for intraperitoneal (i.p.) administration in a volume of 0.1 ml/10 g mouse weight. SS-20

16

(H-Phe-D-Arg-Phe-Lys-NH2) was synthesized as previously described9 and was dissolved

17

in normal saline for continuous or bolus subcutaneous administration.

18

Oxaliplatin-induced peripheral neuropathy model. Oxaliplatin (10mg/kg) was injected



1

intraperitoneally in a volume of 0.1 ml/10 g mouse weight, once per week for 3 weeks. The

2

dose of oxaliplatin administration was chosen according to our previous report.3 Behavioral

3

testing (see below) was performed prior to the first oxaliplatin administration (baseline data)

4

and 5 days after the third oxaliplatin administration.

5

Behavioral testing. The presentation of neuropathic pain was determined by the

6

development of hypersensitivity to mechanical and cold non-noxious stimuli. The

7

mechanical hypersensitivity was assessed by the decrease in paw withdrawal mechanical

8

thresholds using the von Frey test. Mice were placed in a clear plastic chamber with a wire

9

mesh floor, which provided full access to the planter surface of the hind paws. Mice were

10

allowed to habituate for at least 15 min before testing. The paws were touched with one of a

11

series of 10 von Frey filaments (Semmes-Weinstein Monofilaments; Stoelting Co., Wood

12

Dale, IL) with logarithmically incremental stiffness (0.005–3.630 g) starting with the filament

13

of 0.407 g. The 50% mechanical withdrawal thresholds were determined using the up-down

14

method.20 Cold hypersensitivity to non-noxious cold stimulus was assessed by counting the

15

number of forepaw-shaking behavior of mice placed on a cold plate (LHP-1700CP; TECA,

16

Chicago, IL) set at 15℃ (15℃-cold plate test) during a 2.5 min test period. The cold plate

17

test was performed following the von Frey test. Behavioral testing was performed in a

18

blinded manner with respect to all drug administration.


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1

Effect of continuous SS-20 treatment during neuropathy development. In order to

2

examine the effects of continuous administration of SS-20 during repeated oxaliplatin

3

administrations, SS-20 at 5 mg/kg/day, 10 mg/kg/day or vehicle (normal saline) was

4

continuously administered via subcutaneously implanted Alzet Micro-Osmotic Pumps

5

(Model 1004, Alzet, Cupertino, CA; pumping rate of 0.11 µl/h for 28 days). Two pumps were

6

implanted to bilateral sides of the back in each mouse under sevoflurane anesthesia 2 days

7

before the first oxaliplatin injection to allow the infusion rate of the pumps to stabilize. SS-20

8

or vehicle was administered continuously via the pumps until the end of the experiment.

9

Behavioral testing was performed prior to the implantation of the pumps (baseline data) and

10

5 days after the last injection of oxaliplatin.

11

Quantification of IENFs. After the last behavioral testing of mice that were administered

12

SS-20 at 10 mg/kg/day or vehicle, planter skin specimens were excised from the hind paw

13

between the calcaneous and the digital tori under sevoflurane anesthesia. The specimens

14

were immediately soaked in 4% paraformaldehyde for overnight fixation and then

15

transferred to 30% sucrose and left overnight at 4°C. The specimens were then embedded

16

in TissueTek OCT compound (Sakura Finetek Europe B.V., Zouterwoude), frozen and sliced

17

on a cryostat into 25 µm sections. The sections were collected in 0.01 M phosphate buffer

18

saline (PBS) and processed by a free-floating protocol. The sections were incubated in a



1

blocking solution that consisted of 0.5% normal goat serum and 0.3% Triton-X100 in PBS for

2

30 min at room temperature. They were then incubated overnight at 4°C in primary

3

antibodies in the blocking solution. Antibodies against protein gene-product 9.5 (rabbit,

4

affinity purified, Enzo Life Sciences, Farmingdale, NY, USA) were used. Following this step,

5

the sections were incubated in biotinylated goat anti-rabbit IgG (Vector Laboratories,

6

Belmont, CA, USA) solution for 90 min at room temperature, then incubated with Alexa Fluor

7

488 streptavidin conjugate (Molecular Probes, Eugene, OR, USA) for 60 min at room

8

temperature. Between each step, the sections were rinsed with PBS three times. The

9

sections were observed under a fluorescence microscope. The number of IENFs per mm of

10

the epidermal border was counted in 3 random sections and averaged for each mouse.

11

Quantification of IENFs was performed blindly with respect to drug administration.

12

Effect of acute SS-20 treatment on established neuropathic pain. In order to examine

13

the effects of acute administration of SS-20 on established oxaliplatin-induced neuropathic

14

pain symptoms, other mice that had received 3 weekly administrations of oxaliplatin were

15

tested 5 days after the last oxaliplatin administration. A single dose of SS-20 (10 mg/kg) or

16

saline was given subcutaneously to each mouse in a volume of 0.1 ml/10 g mouse weight.

17

Behavioral testing was performed prior to the first oxaliplatin administration (baseline data),

18

and before and 1 h after the administration of SS-20 or saline on the day of the drug test.


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1

Statistical analysis. Statistical analyses were carried out with SigmaPlot statistical

2

software package for Windows (version 13.0; Systat, San Jose, CA). Data were analyzed

3

using one-way analysis of variance (ANOVA) with Bonferroni correction or two-way

4

repeated ANOVA with Bonferroni correction. For all tests, p < 0.05 was considered

5

statistically significant.

6

7

ABBREVIATIONS

8

CIPN, chemotherapy-induced peripheral neuropathy; ROS, reactive oxygen species; IENF,

9

intraepidermal nerve fiber; ATP, adenosine triphosphate

10

11

AUTHOR INFORMATION

12

Corresponding Author

13

*E-mail: [email protected]. Phone: +81-3-3433-1111. FAX: +81-3-5400-1247

14

Author Contributions

15

S.T. and N.S. were involved in experiments and data analysis of the study. H.H.S. was

16

involved in planning of the study. P.W.S. was involved in the synthesis of SS-20 and

17

planning of the study. M.S. conceived the study and was involved in experiments and data

18

analysis.



1

Funding Sources

2

The work was supported by a grant-in-aid for Scientific Research to M.S. (grant no.

3

24592358) from the Japan Society for the Promotion of Science (Tokyo, Japan), and grants

4

to P.W.S. from the U.S. National Institutes of Health (DA004443) and the Canadian

5

Institutes of Health Research (MOP-89716).

6

Conflicts of Interest

7

H.H.S. is the inventor of SS-20. SS-20 has been licensed by the Cornell Research

8

Foundation to Stealth Biotherapeutics for commercial research and development. H.H.S. is

9

the Scientific Founder and has equity ownership in Stealth Biotherapeutics. The other

10

authors have no conflict of interest to declare.

11


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1 2

Figure 1. Chemical structures of SS-31 and SS-20.



1 2

Figure 2. Effects of continuous administration of SS-20 on mechanical hypersensitivity (a)

3

and cold hypersensitivity (b) induced by repeated administrations of oxaliplatin. Mechanical

4

hypersensitivity was assessed by determining paw withdrawal mechanical thresholds using

5

the von Frey hair test. Cold hypersensitivity was assessed by counting the number of

6

forepaw-shaking behavior of mice placed on a cold plate set at 15℃ during a 2.5-min test

7

period. Baseline measurements were made prior to the implantation of Alzet pumps filled

8

with SS-20 at 5 mg/kg/day (n=9), SS-20 at 10 mg/kg/day (n=10) or vehicle (normal saline,

9

n=6). The Alzet pumps were implanted 2 days before the first oxaliplatin injection.

10

Oxaliplatin (10mg/kg) was administered intraperitoneally in mice, once per week for 3 weeks.

11

Post-oxaliplatin measurements were made 5 days after the last administration of oxaliplatin.

12

##

13

group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to baseline of the same group. Data

14

were analyzed by two-way repeated ANOVA followed by the Bonferroni method.

p < 0.01,

###

p < 0.001 compared to the corresponding time point of the vehicle control


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1 2

Figure 3. Changes in intraepidermal nerve fiber density by repeated administrations of

3

oxaliplatin with or without concomitant SS-20 administration. Fluorescence microscopic

4

photograph of IENFs in the planter skin harvested from a naïve mouse (a), a mouse that

5

received

6

administration of vehicle (normal saline) (b), and a mouse that received three weekly

7

administrations of oxaliplatin (10 mg/kg) with continuous administration of SS-20 (10

8

mg/kg/day) (c). Scale bars are 50 µm.

three

weekly

administrations

of

oxaliplatin

9


(10mg/kg)

with

continuous


1 2

Figure 4. Intraepidermal nerve fiber (IENF) densities of plantar skin (hind paw) in naïve

3

mice (n=4), mice given repeated administrations of oxaliplatin with continuous

4

administration of vehicle (normal saline: NS) (Oxaliplatin+NS, n=3), and mice given

5

repeated administrations of oxaliplatin with continuous administration of SS-20 at 10

6

mg/kg/day (Oxaliplatin+SS-20, n=3). Oxaliplatin at 10 mg/kg was administered once a

7

week for 3 weeks. IENFs counts were determined per 1 mm of epidermal border. The skin

8

specimens were harvested 7 days after the last administration of oxaliplatin. Data were

9

analyzed by one-way ANOVA followed by the Bonferroni method.

10


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1 2

Figure 5. Effects of acute administration of SS-20 on mechanical hypersensitivity (a) and

3

cold hypersensitivity (b) induced by repeated administrations of oxaliplatin. Mechanical

4

hypersensitivity was assessed by determining paw withdrawal mechanical thresholds using

5

the von Frey hair test. Cold hypersensitivity was assessed by counting the number of

6

forepaw-shaking behavior of mice placed on a cold plate set at 15℃ during a 2.5-min test

7

period (cold plate test). Baseline measurements were made prior to the first administration

8

of oxaliplatin. Oxaliplatin (10 mg/kg) was administered intraperitoneally in mice, once per

9

week for 3 weeks. Drug tests were performed on the 5th day after the last administration of

10

oxaliplatin. The von Frey tests and the cold plate tests were performed before and 1 h after

11

acute subcutaneous administration of SS-20 (10 mg/kg, n=6) or vehicle (n=5). ** p < 0.01,

12

*** p < 0.001 compared to baseline of the same group. Data were analyzed by two-way

13

repeated ANOVA followed by the Bonferroni method.



For Table of Contents Use Only The protective effect of a mitochondria-targeted peptide against the development of chemotherapy-induced peripheral neuropathy in mice Satoshi Toyama, Naohito Shimoyama, Hazel H. Szeto, Peter W. Schiller, Megumi Shimoyama 80x39mm (300 x 300 DPI)


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Protective Effect of a Mitochondria-Targeted Peptide against the

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