Oregon Health & Science University, Department of...

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*Protection Against Cisplatin-induced Toxicities by N-acetylcysteine and Sodium

Thiosulfate as Assessed at the Molecular, Cellular and in Vivo Levels

D. Thomas Dickey, Y. Jeffrey Wu, Leslie L. Muldoon, and Edward A. Neuwelt

Oregon Health & Science University, Department of Neurology (DTD, YJW, LLM, EAN)

and Department of Veteran’s Affairs (EAN), Portland, OR 97239

JPET Fast Forward. Published on June 10, 2005 as DOI:10.1124/jpet.105.087601

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 10, 2005 as DOI: 10.1124/jpet.105.087601

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Running Title: Chemoprotection against cisplatin toxicities

Correspondence and reprint requests:

Edward A. Neuwelt, MD

L 603 3181 SW Sam Jackson Park Rd

Portland, OR 97239

W: 503-494-5626

F: 503-494-5627

E: [email protected]

# text pages: 12

# of tables: 1

# of figures: 5

# of references: 31

# of words in abstract: 250

# of words in introduction: 556

# of words in discussion: 1313

Abbreviation List:

CDDP cisplatin, cis-diamminedichloroplatinum

NAC N-acetylcysteine

STS sodium thiosulfate

ABR acoustic brainstem response

BUN blood urea nitrogen


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CR creatinine

IV intravenous

SCLC small cell lung cancer

PARP poly (ADP-ribose) polymerase

DME Dulbecco’s Modified Eagle Medium

Recommended section assignment:

Chemotherapy, antibiotics and gene therapy


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ABSTRACT

Cisplatin (CDDP) is a common, highly toxic chemotherapeutic agent. This study

investigates chemoprotective effects of N-acetylcysteine (NAC) and sodium thiosulfate

(STS) on in vitro and in vivo CDDP toxicities. For ototoxicity studies CDDP (6 mg/kg)

was administered to rats via a retrograde carotid artery infusion. Auditory brainstem

response (ABR) thresholds at 4-20 kHz were tested before and 7 days post-treatment.

STS (8 g/m 2 IV) was administered at 4, 8, or 12 hours after CDDP. For nephrotoxicity

studies rats were treated with CDDP intraperitoneally (10 mg/kg) before or after NAC

(400mg/kg) or STS (8g/m2), and blood urea nitrogen (BUN) and creatinine (CR)

concentrations were measured after 3 days. In vitro cytotoxicity and chemoprotection in

human tumor cell lines were assessed by cell viability and immunoblotting assays. Rats

treated with STS 4 h after CDDP exhibited no hearing change. The STS 8 h group had

less otoprotection, while 12 h rats had ototoxicity. CDDP induced high BUN and

creatinine, corresponding to renal tubule toxicities. All NAC-treated animals showed

normal BUN and reduced creatinine levels compared to CDDP alone, and no

histopathological evidence of nephrotoxicity. Delayed STS treatment was not

consistently protective against nephrotoxicity. STS administration fully protected against

the in vitro cytotoxic and apoptotic effects of CDDP if added within 2 h of CDDP, but

chemoprotection decreased if STS administration was 4 h and was minimal by 6 h after

CDDP. Thus, the chemoprotection route and timing of administration can be

manipulated to maintain CDDP anti-tumor efficacy while protecting against toxicities.


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Introduction

Cisplatin (CDDP) is a widely used and effective chemotherapeutic agent that

binds to and alkylates DNA and triggers transcription inhibition, cell cycle arrest, and

apoptosis (Wu et al., 2005; Siddik, 2003). In addition, CDDP generates reactive oxygen

species (ROS), which are known as one of the pathogenic intermediates following

chemotherapy (Masuda et al., 1994). CDDP is dose limited by a high incidence of

toxicities, including progressive and irreversible ototoxicity and nephrotoxicity. Animal

models have demonstrated that the CDDP-induced hearing loss involves loss of the

inner and outer hair cells of the inner ear (Blakley et al., 2002). Ototoxicity has a

significant negative impact on patient quality of life, especially in pediatric cases, where

more than 50% of children develop ototoxicity (Gilmer-Knight et al., 2005, in press).

Even minimal and mild hearing loss in high frequency regions above 2000 Hz

considerably increase a child's risk for academic difficulties, social-emotional problems

(Gilmer-Knight et al., 2005, in press) and increased levels of fatigue in the learning

environment (Bess et al., 1998). Methods to decrease this effect would increase the use

and dosages of the effective platinum-based chemotherapeutic agents.

In the past, nephrotoxicity was a major consequence of CDDP treatment (Jones

and Bassinger, 1989). The kidney accumulates CDDP to a higher degree than any other

organ, resulting in necrosis of the terminal portion of the proximal tubule and apoptosis

in the distal nephron (Arany and Safirstein, 2003). Toxic renal failure induces the

production of ROS, which are responsible for the induction of tubular epithelial cell

death. This is mediated by caspases and endonucleases (Basnakian et al., 2002). An

increased rate of apoptosis has been detected in both human and experimental

glomerular scarring, and CDDP induced apoptosis in cultured human proximal tubular


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epithelial cells (Razzaque et al., 2002 ). While nephrotoxicity can be lessened with

extensive hydration by crystalloid administration, it can still be dose-limiting.

Anti-oxidants such as the endogenous tripeptide glutathione or exogenously

administered thiols can protect against CDDP cytotoxicity in vitro (Wu et al., 2005).

Animal studies (Campbell et al, 1996; Church et al., 1995; Muldoon et al., 2000; Neuwelt

et al., 1996; Dickey et al., 2004) and clinical trials (Doolittle et al., 2001; Neuwelt et al.,

1998; Robbins et al., 2000) have shown that thiosulfates can protect against platinum-

induced ototoxicity. Further, Jones and Bassinger (1989) demonstrated that D-

methionine provided CDDP nephroprotection.

Concerns about diminishing the oncological effects of CDDP have limited the

clinical use of protective agents (Blakley et al., 2002). Muldoon et al. (Muldoon et al.,

2000) reported that delaying sodium thiosulfate (STS) for 8 hours did not adversely

affect its oncological activity but still offered protection from carboplatin-induced

ototoxicity in a guinea pig model. Neuwelt et al. (Neuwelt et al., 2004) demonstrated that

the efficacy of chemotherapy for rat brain tumors was not affected by thiol

chemoprotection. A clinical trial showed otoprotection after carboplatin increased with a

longer delay of STS infusion (Doolittle et al., 2001).

The present study tested the effect of administering NAC or STS on the

incidence and magnitude of CDDP-induced toxicities, using molecular, cellular and in

vivo models. NAC and STS both have about 15 minute plasma half lives, while the initial

plasma half-life of CDDP is 25 to 49 minutes. We evaluated the impact of the timing of

these agents so as to optimize separation of chemoprotection from CDDP anti-tumor

efficacy.


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Methods

Animals. Animal studies were performed in accordance with guidelines established by

the OHSU Institutional Animal Care and Use Committee (IACUC) and with their approval

of the protocols. Animals were housed at the OHSU Animal Facility and under the care

of staff veterinarians.

Pharmocological Agents. Cisplatin (Platinol) was obtained from Bristol-Meyers Squibb

(New York, NY), and N-acetylcysteine sterile solution was obtained from Abbott

Laboratories (North Chicago, IL) via the Oregon Health and Sciences University hospital

pharmacy. Sodium thiosulfate (Sigma-Aldich, St. Louis, MO) was also obtained from the

OHSU pharmacy.

CDDP Administration for the Ototoxicity Study. Long Evans adult female rats were

weighed, induced with isoflurane inhalant (2% + 1.5% O2), intubated, placed on a

respirator and prepped for surgery. Isoflurane was then replaced with propofol (800

µg/kg/min intravenously (IV)) and a 50% nitrous/50% oxygen mixture. A ventral midline

incision was made from mandible to manubrium. The right carotid bifurcation was

exposed and freed, and the right external carotid artery was cannulated. The right

internal carotid artery was clamped and CDDP (6 mg/kg) was infused retrograde through

the external carotid catheter into the aorta using a Harvard Apparatus infusion pump set

at 3 ml/min. The cannula was removed and the skin incisions were closed using 4-0

Vicryl in a simple continuous pattern. Four hours post-CDDP infusion, STS (8 g/m2 IV)

was then given to the treated group, and IV saline was given to the untreated group.


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This procedure was repeated giving STS 8 and 12 hours after CDDP. Health and well-

being of the animals were monitored daily post-surgery.

Evoked potentials. To test for cochlear function, auditory brainstem responses

(ABR’s) were measured in 16 Long Evans adult female rats. Baseline Acoustic

Brainstem Responses (ABR) were measured before the administration of CDDP and

were repeated 7 days after CDDP with or without NAC or STS. All recordings were

conducted with the animal in a double walled audiometric booth (Acoustic Systems). The

animals were anesthetized prior to the ABR measurement with ketamine (60-120mg/kg)

delivered intraperitoneally (IP). ABR’s were recorded using the Intelligent Hearing

Systems Smart EP Evoked Potentials system, version 3.30.Platinum subdermal needle

electrodes (Grass Instruments) were placed at each mastoid process, with a ground

electrode at the forehead. Stimuli were presented through an EAR 3A insert earphone,

coupled to the HIS HFT9810-016 high frequency transducer, placed into the external

auditory canal of the animal. Thresholds were measured in response to tonebursts

centered at frequencies 4, 8, 12, 16 and 20 kHz. Stimuli were gated by a Blackman

envelope with a 2 msec rise/fall time and a 0 ms plateau. Stimuli were presented at a

rate of 19.3 cycles per second. Each recording was an average of 500 to 1000

individual responses. An intensity series was obtained for each animal beginning at 75

dB SPL and proceeding in 10 dB decrements to below threshold. Threshold was

defined as the lowest intensity capable of eliciting a replicable, visually detectable

response (Campbell et al., 1996).

CDDP Administration for Nephrotoxicity. To test for CDDP nephrotoxicity protection,

rats were anesthetized with isoflurane and given CDDP 10 mg/kg IP, since the ototoxic

dose of CDDP 6 mg/kg IA did not result in consistent nephrotoxicity, 15 minutes after IV


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infusion of either saline or NAC (400 mg/kg). Other groups of rats were given STS (8

g/m2 IV) 4, 8, or 12 hours after CDDP 10 mg/kg IP. Three days after treatment, the

animals were weighed and 0.1 ml blood samples taken for blood chemistry analysis of

blood urea nitrogen (BUN) and creatinine (CR) using an i-Stat Portable Clinical Analyzer

(Heska Corp. Ft. Collins, CO). Changes in BUN and CR were used to indicate

differences in CDDP-induced nephrotoxicity between the NAC and saline-treated rats.

Some samples were subsequently subjected to pathological analysis and the results

compared to the blood values. Rats were sacrificed using an intracardiac injection of

pentobarbitol, and the kidneys were resected and fixed by immersion in formalin for at

least three days. Tissues were processed by ARUP Laboratories (Salt Lake City, UT)

with 5 µm sections stained with H&E. Kidney sections were analyzed by Lawrence D.

McGill, DVM, PhD of ARUP Animal Division.

Cell culture and in vitro analyses. The B.5 LX-1 small cell lung carcinoma (SCLC) cell

line was maintained as a free-floating cell suspension in spinner flasks, in medium

RPMI-1640 supplemented with 12% fetal bovine serum (Irvine Scientific, Santa Anna

CA) plus gentamycin, penicillin, and streptomycin. DAOY medulloblastoma cells,

obtained from American Type Culture Collection (Rockville, MD), were cultured in

Minimal Essential Medium supplemented with 10% serum and antibiotics. The SKOV3

ovarian cancer cells and U87MG glioblastoma cells, obtained from Dr. Gail Clinton, were

cultured in Dulbecco’s Modified Eagle Medium (DME) supplemented with 10% fetal

bovine serum and antibiotics. Rat fibroblasts (Rat1), obtained from Dr. Bruce Magun at

OHSU, were cultured in DME with 5% serum and antibiotics. Live cell number was

determined with the WST-1 Cell Proliferation Assay Kit from Chemicon International Inc

(Temecula CA), as previously described (Wu et al., 2005). STS was added 0, 2, 4, 6, or


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8 h after CDDP (30-50 µM) in 4 wells per condition, repeated 2 times in each cell type,

and viable cell number was determined 44-48 h after CDDP addition.

Western blot analysis was performed as previously described (Wu et al., 2005).

Both human lung carcinoma (LX-1 SCLC cells) and Rat1 fibroblasts were tested. Rabbit

anti-poly (ADP-ribose) polymerase (PARP) antibody was obtained from Cell Signalling

Technology (Beverly, MA) and anti-tubulin antibody from Sigma (St. Louis, MO).

Statistics. Means and standard errors were determined using Microsoft Excel

software or Graphpad Prism 4 software. Statistical differences between groups were

determined using an Anova for repeated measures or Student’s t-test using Microsoft

Excel software or Graphpad Prism 4 (San Diego, CA) software.

Results

Ototoxicity. CDDP induces ototoxicity in rats if given intra-arterially so that the

vertebral arteries are perfused, at doses less than other routes of administration (Dickey

et al., 2004). Mean hearing thresholds following CDDP treatment for the saline- and

STS-treated groups are shown in Fig. 1a-c. STS was otoprotective in a time-dependent

manner. STS was significantly protective (p<0.05) against CDDP-induced ototoxicity if

given 4 hours post-CDDP (Fig. 1a), similar to that found with NAC given 15 minutes prior

to CDDP (Dickey et al., 2004). In the control group, the differences between pre- and

post-treatment hearing thresholds were statistically significant at each frequency. In the

8 hour STS group, no change from baseline hearing thresholds was found at low

frequencies. Some ototoxicity was found at higher frequencies, but significant protection

(p<0.05) was found compared to saline controls (Fig. 1b). The treatment by time

interaction indicates there is a significant pre- vs. post-treatment difference for the STS


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12 hour post-CDDP group at every frequency, indicating no STS protection at this time

point (Fig. 1c).

Nephrotoxicity. As shown in Fig. 2a, the 15 minute prior to CDDP NAC-treated

rats had a normal BUN (mean=23.6 mg/dL; p<0.001) 3 days after the IP model of CDDP

administration, while the CDDP+saline treated rats had abnormally high BUN

(mean=130.8 mg/dL). The 30 minute-prior and 4 hour-post NAC rats also had

significantly lower BUN than the CDDP alone group (p<0.01). Also, data in Fig. 2b

indicates that the 15 minute NAC prior to CDDP animals had normal CR levels

(mean=0.8 mg/dL), while the CR levels in the CDDP+saline-treated rats were

abnormally elevated (mean=8.2 mg/dL; p<0.001). The NAC 30 minutes prior and 4

hours post CDDP also had significantly lower CR (p<0.001). The STS-treated rats were

provided with no consistent protection against renal toxicity, as indicated by high BUN

(Fig. 2c), although the data for the STS 4-hour post-CDDP group was mixed, with two

individuals showing nephroprotection.

Table 1 presents the correlation of pathological interpretation of CDDP-treated

kidneys with BUN results. There is a direct correlation between a high level of BUN and

the presence of renal tubular damage found in histological samples of kidneys from the

same animal. The pathologist was blinded as to treatment and evaluated the kidney

sections at random for the presence or absence of lesions with no regard to

quantification. Kidneys from rats with high BUN showed varied severities of acute

tubular degeneration. Inflammation was minimal. Occasional rats demonstrated more

severe degeneration along the cortical medullary junction with calcification. Proteinuria in

medullary tubules commonly accompanied tubular degeneration. These kidneys were

severely damaged. Rats with normal BUN showed no significant lesions in the sections


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of kidneys. Fig 3 shows photomicrographs of representative kidney sections of rats with

(A) and without (B) CDDP-induced renal tubular damage.

Cytotoxicity. The time dependence for STS chemoprotection was evaluated to

determine how long the addition of chemoprotectant could be delayed after CDDP

treatment and remain effective against cytotoxicity (Fig. 4). Incubation for 48 hours with

CDDP (17 µM) reduced cell viability by 57.6 ± 3.5 % in U87 glioblastoma cells, 80.8 ±

3.7 % in SKOV3 ovarian carcinoma cells, and approximately 100% in both DAOY

medulloblastoma cells and B.5 LX-1 SCLC cells. STS (2 mg/ml) was protective against

CDDP-induced cytotoxicity when added either concurrent with CDDP or up to 2 hours

after CDDP. Delayed administration of STS reduced its protective activity against CDDP

cytotoxicity. If STS was administered 4 h after CDDP, its protective activity was reduced

to 30-40% of the maximal protection seen with concurrent administration, which was a

significant decrease in all cell lines, but remained significantly chemoprotective

compared to no STS at all. When delayed until 6 h post CDDP, STS showed no

significant chemoprotective activity in any cell type tested. The magnitude of STS

chemoprotection was dependent on CDDP concentration, particularly in the 2-4 hour

window (Fig. 4b).

Western Blot analysis of signaling and apoptosis. To compare the difference of

STS and NAC chemoprotective activity, we found that STS has slightly better protective

effect against CDDP-induced apoptosis (Figure 4). At the 0.25 mg/ml. level, STS was

completely protective against CDDP-induced cell death indicated by the presence of

cleaved PARP protein. Both NAC and STS reverse CDDP stimulated phosphorylation of

Extracellular Signal-Regulated Kinase (Wu et al., 2005).


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For both human lung carcinoma (LX-1 SCLC) and Rat 1 fibroblast cell types,

STS completely blocked CDDP-induced apoptosis when added within 1 h of CDDP

treatment. PARP cleavage was apparent if NAC/STS administration was delayed until 2

h or later after CDDP treatment, although it appeared there was partial protection even

when STS was delayed until 8 h after CDDP. After this time point, no protection was

evident (Figure 5). These data suggested that the presence of STS in the culture

medium is required to have anti-apoptotic effect.

Discussion

Platinum-based chemotherapy is currently the most effective form of

chemotherapy for a variety of malignant tumors, including head and neck cancer

(Blakley et al., 2002) and several pediatric malignancies. Ototoxicity is the primary dose

limiting factor in CDDP therapy (Blumenreich et al., 1985) that both reduces quality of life

and restricts treatment protocols. CDDP-induced nephrotoxicity can be alleviated with

hydration using crystalloid therapy but can still be dose limiting. This study evaluated

molecular, in vitro, and in vivo chemoprotection against CDDP toxicities with the thiol

STS.

Ototoxicity and protection

CDDP is known to cause loss of hearing by progressive destruction of outer hair

cells of the cochlea. The damage progresses from basal to apical and from outer to inner

hair cells (Blakley et al., 2002). High frequencies are usually affected before low

frequencies in humans who receive CDDP because the cochlea is tonotopically

arranged. The model of CDDP-induced ototoxicity in this study utilized an aortic delivery

of CDDP, first reported by Dickey et al. (Dickey et al., 2004), and uses clinically relevant


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doses of CDDP (20-80 mg/m2 is equivalent to 2.9-11.9 mg/kg in a 250g rat), lower than

the 15 mg/kg dose used in other studies (Campbell et al., 1996).

This study presents evidence that 4 hour post-treatment with STS IV can prevent

CDDP-induced ototoxicity, as indicated by changes in ABR threshold. STS delivered 8

hours after CDDP provided less protection, all at the higher frequencies, while STS

given after 12 hours provided no otoprotection. Previous studies in this lab found

corresponding protection by NAC given in this fashion for both ototoxicity and systemic

toxicity as indicated by weight loss (Dickey et al., 2004).

Most thiols are electrophilic and are thought to act as free radical scavengers.

The mechanism of protection of STS, and perhaps NAC, may also be due to covalent

binding of the molecule to the platinum, producing an inactive complex (Neuwelt et al.,

2001; Fuertes and Castilla, 2003). Schweitzer (Schweitzer, 1993) showed that sulfur-

containing compounds may prevent CDDP from interacting with target molecules,

displacing platinum after it is bound. D-methionine has been shown to protect against

CDDP-induced ototoxicity (Campbell et al., 1996). NAC was shown to be protective in

vitro of outer-hair cells taken from guinea pigs (Feghali et al., 2001). Chemoprotection

against platinum-induced otoxicity was found in a clinical trial using delayed high-dose

thiols for hearing protection (Doolittle et al., 2001). With STS otoprotection, there is no

differential protective effect in normal cells vs. tumor cells as occurs with amifostine.

Since there is no selective prevention of CDDP effects in hearing cells compared to

tumor cells, the issue then becomes delivery and timing.

Nephrotoxicity and protection

We tested the hypothesis that pretreatment with the thiol agent NAC or post-

treatment with STS would reduce renal toxicity of CDDP. CDDP induced nephrotoxicity


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in rats, shown by significant and abnormally high changes in BUN and CR. Rats that

received NAC prior to CDDP had normal BUN values (Fig 2a,b). Post-treatment with

STS showed no consistent renal protection, although the 4 hour post-CDDP treatment

protected some animals (Fig 2c).

As we have demonstrated with the data in Table 1, the BUN and CR levels

correlate well with renal tubule damage seen in histological sections. A similar

correlation between kidney histology and high BUN and CR levels after a single dose of

CDDP was reported by Yildirim et al. (2003). NAC also protected against weight loss,

which can be an indication of renal damage (Zhang et al., 1999; Ammer et al., 1993).

Timing of Thiols for chemoprotection

Our in vitro studies show that STS was protective against CDDP-induced

cytotoxicity when added either concurrently or 2 hours after CDDP. The protective in

vitro effect diminished after 4 hours, and was gone by 6 hours. Data for in vivo oto-

protection of STS (Fig. 1c) show a similar reduction in effect if administered 8 hours or

later after CDDP.

CDDP induces apoptosis via activation of caspase 3 (Wu et al., 2005; Siddik,

2003; Ludwig and Oberleithner, 2004). Caspase 3 can be activated by caspase 9, which

is activated by the release of cytochrome c from the mitochondria (Schuler et al., 2000;

Zhan et al., 1999). STS blocks the apoptotic pathways induced by CDDP if given within

2-4 hours of CDDP, but the protective effect wanes after 8 hours and is not present after

12 hours (Fig 5). This is indicated by the presence of cleaved PARP protein, and is

similar to previous results in our lab with NAC (Wu et al., 2005). The timing of apoptosis

is reflected in the results for oto- and nephro-protection of delayed STS. STS given 4

hours after CDDP was otoprotective, less so after 8 hours, and non-protective if given 12

hours after CDDP (Fig 1c). The renal data shows limited protection of the tubules by


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STS given 4 hours after CDDP and no protection 8 or 12 hours (Fig 2c). The thiols are

also precursors of L-cysteine and the glutathione pathway, and act as potent scavengers

of free radicals (Zafarullah et al., 2003). Scavenging of ROS protects tubular epithelium

from caspase activation and from cell death (Basnakian et al., 2002). The timing of

CDDP administration after NAC becomes an issue, due to the rapid clearance of NAC

(Neuwelt et al., 2001). Glutathione protection against CDDP-induced nephrotoxicity was

found to be critically dependent on timing of thiol administration (Zunino et al., 1989).

The issue of potential interactions of chemoprotection with chemotherapy

efficacy is of concern to all oncologists. Previous studies in this laboratory demonstrated

that delayed STS administration after treatment with CDDP reduced ototoxicity in guinea

pigs, at times and concentrations which did not reduce antitumor activity (Muldoon et al.,

2000). In a second study (Neuwelt et al., 2004) we assessed STS and NAC in an

intracerebral tumor model. Chemotherapy was delivered to the LX-1 small cell lung

carcinoma brain tumor xenografts with carotid artery infusion and BBB disruption, either

before or after treatment with NAC and/or STS. We found that delayed administration of

STS, 4 and 8 hours after chemotherapy, did not alter anti-tumor efficacy against the

intracerebral tumor model (Neuwelt et al., 2004). Delayed STS had no impact on the

tumors either alone or in combination with NAC pretreatment, even though bone marrow

toxicity was significantly reduced (Neuwelt et al., 2004). These antitumor studies were

done using carboplatin (Diammine(cyclobutane-1,1-dicarboxylato(2-)-O,O')platinum),

which may have less efficacy than CDDP against some tumors, but also has fewer non-

hematological dose-limiting toxicities. The current study focuses on CDDP, which is

widely used in both adult and pediatric oncology. The reduction of the dose-limiting

toxicities of CDDP would be a benefit in future clinical trials.


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In summary, delayed administration of STS provides protection against CDDP-

induced toxicities in vitro and in the in vivo ototoxicity rat model. NAC provides

protection in the nephrotoxicity model. Current studies suggest the efficacy of CDDP

therapy can be maintained with NAC and STS protection by separating the thiols from

platinum in time and space (Wu et al., 2005; Muldoon et al., 2000; Doolittle et al., 2001;

Neuwelt et al., 2004). Clinical trials using STS protection for brain tumor patients

undergoing carboplatin chemotherapy after blood-brain barrier opening show that

increasing the delay of STS administration from 2 to 4 hours improved otoprotection,

perhaps because it increased the STS to carboplatin ratio. As a result there was a

marked decrease in the need for hearing aids (Doolittle et al., 2001). The current

results, together with previous in vivo tumor efficacy studies (Muldoon et al., 2000;

Neuwelt et al., 2004) suggest that chemoprotection route and timing of administration

can be manipulated to maintain CDDP anti-tumor efficacy while protecting against

chemotherapy toxic side effects. As these studies utilized carboplatin instead of CDDP

as in the current study, further in vivo experiments are necessary and underway to affirm

the anti-tumor efficacy of CDDP in this model. These data would lead to considerations

for further clinical trials.

Acknowledgments:

Editing assistance was provided by D. Michelle Judd of The Publishing Doctor. Expert

technical support by Shannan Walker-Rosenfeld, Kristina Pinkston and Kristin Gilmer-

Knight was greatly appreciated.


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Footnotes:

* This work was supported by a Veteran’s Administration Merit Review Grant and by NS

33618 from the National Institute of Neurological Disorders and Stroke to EAN.

Conflict of interest statement: Dr. Neuwelt, Dr. Muldoon, and the Oregon Health &

Science University have significant financial interests in Adherex Technologies, Inc., a

company that may have a commercial interest in the results of this research and

technology. The potential conflict of interest has been reviewed and a management plan

approved by the Oregon Health & Science University Conflict of Interest in Research

Committee has been implemented.

Correspondence and reprint requests:

Edward A. Neuwelt, MD

L 603 3181 SW Sam Jackson Park Rd

Portland, OR 97239

W: 503-494-5626

F: 503-494-5627

E: [email protected]


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Legends for Figures

Figure 1. Effect of STS post-treatment on ototoxicity. Normal Long Evans female rats

received saline or STS 8 g/m2 IV post-administration of CDDP 6mg/kg by the intra-

arterial technique. ABR thresholds were obtained prior to treatment and 7 days after

CDDP. The data are shown as the mean change from baseline of hearing threshold

measured in decibels, averaging both ears.

a. Effect of STS 4 hour post-CDDP treatment on ototoxicity 7 days after CDDP.

b. Effect of STS 8 hour post-CDDP treatment on ototoxicity 7 days after CDDP.

c. Effect of STS 12 hour post-CDDP treatment on ototoxicity 7 days after CDDP.

Figure 2 Effect of thiols on CDDP-induced nephrotoxicity. Long Evans rats were treated

with CDDP 10 mg/kg IP, with or without addition of saline or thiols. BUN and creatinine

were measured 3 days post-treatment. The data are shown as mean SEM.

a. Effect of NAC before CDDP on BUN. Rats received saline or NAC 400 mg/kg

IV 15 or 30 min prior to or 4 hours after treatment with CDDP. Control rats received no

treatment.

b. Effect of NAC before CDDP on Creatinine. Rats received saline or NAC 400

mg/kg IV 15 or 30 min prior to or 4 hours after treatment with CDDP.

c. Effect of STS after CDDP on BUN. Rats received saline or STS 8g/m2 IV 4, 8,

or 12 hours after CDDP.


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Figure 3. Photomicrographs of kidney sections from Long-Evans rats 3 days post-

treatment with CDDP 10 mg/kg IP with or without protection from NAC 400 mg/kg IV.

Hematoxylin and eosin, original magnification: 100X.

a. The renal cortex from the unprotected (no NAC) animals contained zones

such as this in which the proximal tubules have undergone recent necrosis.

Cells have sloughed from the basement membranes in some tubules, and

necrotic debris has accumulated in the lumina. Regenerating cells with

amphiphilic cytoplasm and large nuclei have begun to reline some tubules.

Groups of normal proximal tubules are evident in adjacent areas.

b. As exemplified by this photograph, the kidneys of the NAC-protected animals

contained no evidence of tubular necrosis. Proximal tubules are intact, lined

by histologically normal epithelium.

Figure 4. Time course for STS chemoprotection in vitro. 4a. B5 LX-1 SCLC cells (open

circles), SKOV3 ovarian carcinoma cell (inverted triangles), and U87 glioblastoma cells

(triangles) were treated with 30-50 µM CDDP, with or without addition of STS at the

indicated times. 4b. DAOY medulloblastoma cell were treated with 17 µM or 34 µM

CDDP, with or without STS. Live cells remaining, compared with untreated controls,

were determined 2 days after drug addition, using the WST-1 kit. Data were normalized

to the maximum amount of chemoprotection found with STS given concurrently with

CDDP.

Figure 5. In vitro kinetics of STS chemoprotection against CDDP

toxicity. A) LX-1 SCLC cells were treated with or without 50 µM CDDP B)

Rat1 fibroblast cells were treated with or without 17 µM CDDP. STS (1

mg/ml) was added at the indicated time after CDDP incubation. Whole cell


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lysates were collected after 24 h CDDP treatment, subjected to 7.5%

SDS/PAGE and immunoblotted with PARP and tubulin (loading control)

antibodies.


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Table 1

Correlation of BUN and CR Levels with Pathology Results in Rats after CDDP±NAC

RAT# Ears130 Ears133 Ears167 Ears155 Ears164 Ears132 Ears138 K11 Treatment saline saline saline saline NAC NAC NAC NAC BUN >140 >140 >140 >140 32 26 25 36 CR 7.0 6.7 6.9 7.2 0.8 1.7 0.8 1.1 % WT. Lost -26.2 -26.8 -30.0 -23.8 -7.0 -15.4 -3.3 -9.8 Renal Tubule YES YES YES YES NO NO NO NO Damage (path) Long-Evans female rats were administered 10 mg/kg IP cisplatin ± N-acetylcysteine (NAC) 400 mg/kg IV. Results were taken 3 days post-cisplatin. BUN=blood urea nitrogen mg/dL; CR=creatinine mg/dL.

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