A Novel Non-Toxic Camptothecin Formulation For_Lehnert4novelnontoxic

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Biomaterials 26 (2005) 21152120

A novel non-toxic camptothecin formulation for

cancer chemotherapy

M. Berradaa, A. Serreqia, F Dabbarha, A. Owusub, A. Guptaa, S. Lehnertb,*aBio Syntech Canada Inc, 475 Armand-Frappier, Laval, Que., Canada H7V 4B3

bDepartment of Radiation Oncology, McGill University, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Que., Canada H3C 1A4

Received 15 January 2004; accepted 4 June 2004

Available online 8 September 2004

Abstract

The use of a novel injectable biocompatible and biodegradable camptothecin-polymer implant for sustained intra-tumoral release

of high concentrations of camptothecin is described. The drug delivery vehicle is an in situ thermogelling formulation, which is based

on the natural biopolymer chitosan. This formulation, containing homogeneously dispersed camptothecin, was implanted intra-

tumorally into a sub-cutaneous mouse tumor model (RIF-l). The effectiveness of treatment was measured in terms of tumor growth

delay (TGD). Animals treated with the polymer implants containing camptothecin had significantly longer TGDs compared to

untreated animals as well as to animals treated systemically with camptothecin by intra-peritoneal injection with no evidence of

toxicity in terms of loss of body weight. The results indicate that this novel biodegradable polymer implant is an effective vehicle for

the sustained intra-tumoral delivery of camptothecin which might also be suitable to deliver other insoluble anti-cancer drugs such

as taxol.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Drug delivery; Thermally responsive material; Biodegradation; Intra-tumoral; Chitosan; BST-gel

1. Introduction

Camptothecin is an inhibitor of the DNA-replicating

enzyme topoisomerase I [1] which is believed to act by

stabilizing a topoisomerase I-induced single strand

break in the phosphodiester backbone of DNA, thereby

preventing religation[2,3]. This leads to the production

of a double-strand DNA break during replication,

which results in cell death if not repaired. The naturally

occurring alkaloid was first isolated from the tree

Camptotheca acuminata in China in 1966 [4]. In pre-

clinical studies camptothecin, has been shown to be

effective against human xenografts of colon, lung,

breast, ovarian, and melanoma cancers [57]. But in

spite of the promise demonstrated at the pre-clinical

level, clinical trials were abandoned due to unexpected

toxicity and low antineoplastic activity [810]. In

addition, camptothecin was felt to have limited clinical

potential because of its low solubility and it has been

proposed that local delivery of camptothecin would be a

means to achieve effective drug concentrations in brain

tumors without the undesirable side effects associated

with systemic delivery[11,12].

The therapeutic potential of an intra-tumoral system

for delivery of camptothecin was investigated in Fischer

rats with intra-cranially implanted 9L gliosarcoma. In

this model systemic administration of camptothecin did

not extend survival beyond that of controls, however

intra-tumoral implant of pCPP-SA wafers containing

50% camptothecin (w/w) resulted in significantly

extended survival compared with control group

(po0:001) with 4/10 rats surviving longer than 120

days. Survival was also significantly longer than that

seen in a group given intra-tumoral BCNU (3.8% w/w)

(po0:001). Results obtained with biodegradable poly-

mers in this and other studies are promising, however

these devices have the disadvantage that insertion

requires surgical intervention. In some cases this can

be done when the tumor is resected during the course of

conventional treatment. Nevertheless, dependence on an

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*Corresponding author. Tel.: +1-514-934-1934-44161; fax: +1-514-

934-8220.

E-mail address: [email protected] (S. Lehnert).

0142-9612/$- see front matter r 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biomaterials.2004.06.013


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invasive procedure remains a drawback. Another mode

of drug delivery, biodegradable microspheres avoids the

need for surgery for insertion since they can be

introduced by intra-tumoral injection[13,14]. However,

microspheres do not form a continuous film or solid

implant with the structural integrity needed for certain

prostheses and they may be poorly retained undercertain circumstances because of their small size,

discontinuous nature and lack of adhesiveness.

During the last decade, injectable in situ gel-forming

systems have received increased interest in drug delivery

and tissue engineering. These devices can overcome

many of the problems associated with polymers or

microspheres in that they are both injectable and

produce solid biodegradable implants with a range of

mechanical characteristics in terms of rigidity and load

bearing making them compatible with both soft and

hard tissues. In the present work, we have used a

chitosan polymer to formulate a biodegradable and

biocompatible implant for controlled delivery of camp-

tothecin in a slow-release manner directly into a mouse

fibrosarcoma (RIF-l) implanted subcutaneously in C3H

mice. In this paper, we report the in vitro release

characteristics of the camptothecin-polymer implant

and the in vivo effect of delivering camptothecin in

high concentrations to a murine tumor. The delivery

vehicle used is one of a family of thermosensitive

chitosan solutions, formulated at physiological pH,

which remain liquid at low temperature and turn into

gel when heated. The polymeric matrix used in this

study consists of chitosan polymer and b-glyceropho-

sphate. Addition of glycerol-2-phosphate (b-GP) tochitosan solution produces a hydrogel which undergoes

solgel transition at a temperature close to 37C,

making the formulation a suitable vehicle for drug

administration since the hydrogel when implanted into

the body, flows to fill voids or cavities and becomes solid

at body temperature. These hydrogels are suitable

carriers for water-insoluble drugs and they are non-

toxic and highly biocompatible [1517]. Chitosan is an

important natural polymer widely used for medical and

pharmaceutical applications[18].

2. Materials and methods

2.1. Materials

Chitosan (Deacetylation degree DDA. = 85% deter-

mined by 1H NMR, Mw=3 105 Da) was obtained

from shells of shrimps or lobsters as described elsewhere

[15]. Chitosan flakes were dissolved in aqueous hydro-

chloric acid (0.1n), filtered, dialyzed and precipitated

with aqueous NaOH (6n). The precipitated chitosan

was washed several times with water and vacuum

dried. The white chitosan powder obtained was stored

in a closed flask until used. The chitosan was ultra-

pure with no endotoxins, proteins, inorganics and

heavy metals. Research grade camptothecin (CPT) and

b-glycerophosphate (b-GP) were obtained from Sigma

Chemicals.

2.2. Preparation of an autogelling chitosan solution :chitosan/GP

Chitosan solutions (1.7%w/w) were prepared in 0.1m

hydrochloric acid at room temperature. The chitosan

powders were progressively added to the solvent with

stirring and mixture was stirred for a further 3 h. Sterile

formulations were obtained by autoclaving (121C,

20 min)[16]. To 9 ml of cooled chitosan solution, chilled

45% (w/w) b-GP aqueous solution (sterilized through a

0.20mm filter) was carefully added dropwise to obtain

clear and homogeneous liquid solutions in a final

volume of 10 ml. This formulation contained 1.53%

chitosan, 4.5% b-glycerophosphate. This ratio of

chitosan:GP was similar to that previously described

[17]and had a thermogelling temperature of 37C. The

final solutions were mixed an additional 10 min at 4C.

The pH of the final cold solutions ranged from 6.9 to

7.2. This clear, autogelling system is proprietary and

patented by BioSyntech[17].

2.3. Preparation of chitosan/GP loaded with

camptothecin : chitosan/GP/CPT

Homogeneous clear chitosan/hydrochloric acid solu-

tions (1.7% w/w) were prepared, then autoclaved for20 min at 120C. Chitosan/CPT formulations were

prepared at room temperature by homogeneously

dispersing the powdered camptothecin in chitosan

solutions at a loading of 4.5%w/w under aseptic

conditions. Camptothecin was sterilized by g-irradia-

tion with 25kGy from a 60Co source (MDS Nordion

Inc. Laval, Qc, Canada). Stability of camptothecin

after gamma irradiation was confirmed by HPLC

(procedure described in Section 2.7 below). Irradiated

camptothecin was stored at 4C along with a control

sample of unirradiated powder for up to 2 months.

HPLC analysis on non-irradiated and irradiated sam-

ples gave identical results indicating that gamma

irradiation and storage had not caused any degradation

of the drug. b-glycerophosphate was dissolved in

distilled water and sterilized by filtration through

0.20mm filter. The (b-GP solution was added slowly to

the cooled camptothecin/chitosan dispersion under

aseptic conditions.

2.4. Invitro release study

Four hundred and eighty milligrams of fine camp-

tothecin powder, was dispersed in a 10 ml chitosan

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solution and intimately mixed by stirring (4.5% w/w

loading). Immediately after the addition of b-GP

solution, the mixture was quickly transferred to a mold

to form gels with dimensions 5 15 15mm3. The mold

was pre-coated with a polyethylene glycol solution to

facilitate removal after gelation. After an incubation

time of l h 30 min at 37

C, the gels were removed fromthe mold. In this way a matrix containing homoge-

neously distributed drug was obtained.

In vitro release was performed under infinite sink

conditions using the molded gel immersed at 37C in

500 ml of phosphate buffer pH = 7.4 containing 0.6%

Tween 20. The dissolution system was shaken at

100 rpm. Samples were removed periodically and the

medium was replenished. Released drug was measured

by an HPLC analytical method.

2.5. Cells and tumors

RIF-1 cells were obtained from Dr. Richard Hill

(Ontario Cancer Institute) and were passaged using

standard tissue culture techniques in RPMI 1640 media

supplemented with 10% fetal bovine serum and 1%

antibiotics (all supplied by Gibco BRL). Cells were

trypsinized, collected by centrifugation and resuspended

in media (4 106 cells/ml) before being injected (50 ml)

subcutaneously into the backs of previously shaved

C3H mice. Tumors appeared within 10 days and

reached a volume of 94130mm3 within 3 weeks.

Tumor volumes were calculated from measurements

taken at three orthogonal angles using the formula

(abcp/6).

2.6. Treatment

Treatments were begun when the tumors reached a

volume of approximately 100 mm3. Tumor-bearing

adult female mice (20 g) were separated into 4 experi-

mental groups (n 627) for the different treatments.

One group was an untreated control. Two groups were

injected intratumorally with chitosan/GP or with

chitosan/GP loaded with camptothecin. 10ml of chit-

osan/GP or chitosan/GP/CPT solutions were injected at

room temperature using a 26 G needle inserted in the

center of the tumor. After injection, the needle was held

in place for 34 s before being withdrawn to prevent the

hydrogel from leaking out of the injection site. The

amount of camptothecin incorporated in the hydrogel

was such that the total dose administered was 24 mg/kg.

In the last experimental group, the mice were injected

intra-peritoneally with 50 ml of camptothecin to give a

dose of 60 mg/kg. Camptothecin was dissolved for

injection a mixture of 8.3% Cremophor EL/8.3%

ethanol in 0.75% saline. Tumor measurements were

made daily, and the mice were sacrificed when the end-

point (4 initial tumor volume) was reached. All

animal procedures were conducted according to the

guidelines of the McGill University Animal Care

Committee.

2.7. Measurement of camptothecin by HPLC

Quantitative analysis was performed on a Hewlett

Packard (Series 1100) chromatographic system equipped

with an Autosampler, a solvent module, a UV Detector,

and a System HP ChemStations system. The column

was a reverse-phase Lichrosphere RP18 (Chromato-

graphic Specialities Inc.) column, (particle size 5 mm,

4 250 mm). The HPLC system was eluted isocratically

with methanol: water (63:37; v/v) at room temperature.

The flow rate of the mobile phase was 1.0ml/min and

samples were measured at a wavelength of 370 nm. A

standard curve was constructed by plotting peak area

against concentration. The assay was found to be highly

accurate and reproducible, with a coefficient of deter-

mination = 0.9999.

3. Results

3.1. In vitro release

Chitosan/GP was loaded with camptothecin 4.5%

(w/w) and triplicate samples of polymer gels were

incubated in phosphate-buffered saline solutions con-

taining 0.6% of Tween 20, pH 7.4, 37

C. At intervals,the supernatant fractions were removed and the

medium replenished to maintain the sink conditions.

The amount of drug in the supernatant samples was

quantified by HPLC and the cumulative percentage of

the loaded drug released in the supernatant fractions

was plotted versus time. The amount of drug loaded

initially in the polymer was confirmed by extraction of

the polymer with methanol to release the residual

camptothecin.

The cumulative release of camptothecin versus time of

incubation is shown inFig. 1. Eighty percent of the drug

was released from the implant over 30 days in buffer

containing 0.6% of Tween 20. Approximately 13% was

released in the first 72 h. The drug release from the

formulated chitosan/GP gel was nearly linear under

infinite sink conditions, indicating almost zero-order

release kinetics in the first four weeks after an initial

burst of less than 5% in the first day. The drug

remaining in the chitosan/GP which had been immersed

in buffer for 4 weeks was extracted with methanol and

when this amount was combined with that released over

the preceding 4 weeks it appeared that approximately

80% of intact drug loaded in the gels had been

recovered.

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3.2. Tumor treatment studies

To evaluate its antitumor efficacy camptothecin

formulated in chitosan/GP, was injected intratumorally

using a RIF-1 mouse tumor model. The RIF-1 tumor

has proven to be a useful model for preliminary

screening of various compounds for efficacy because of

its reproducible growth, non-immunogenicity in the

syngeneic host and low frequency of spontaneous

metastases. It was only weakly responsive to camptothe-

cin administered by intra-peritoneal injection (Table 1).

The effect of the camptothecin containing biodegrad-

able polymer implants on tumor growth delay (TGD)

was examined. The results of these studies are shown in

Table 1andFig. 2. The implanted hydrogel containing

4.5% camptothecin by weight was found to be more

effective than systemically delivered camptothecin in

delaying tumor growth (TGDs of 25 and 8 days,

respectively). Tumors injected with blank chitosan/GP

showed no inhibition of growth and had a similar TGD

(7 days) as untreated tumors, confirming that the

hydrogel alone has no effect on the growth of this

tumor. The greater effectiveness of the implant is

presumably due to slow release of the drug in the tumor

and the exposure of tumor cells to toxic drug

concentrations for a prolonged period of time which

causes more cell death than does the short drug

exposure resulting from systemic administration.

Toxicity of camptothecin was evaluated in C3 H mice

with the RIF-1 tumor on the basis of weight loss. None

of the experimental groups showed any significant effect

of treatment on body weight as is shown inFig. 3. No

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Fig. 1. In vitro release profiles of BST-gel loaded with camptothecin,

4.5% by weight. BST-gel/CPT immersed in phosphate buffer pH =

7.4 containing 0.6% of Tween 20.

Fig. 2. Delay of RIF-1 tumor growth after intratumoral (24 mg/kg)

and intraperitoneal (6 mg/kg) injections of camptothecin. OO Notreatment, Blank BST-gel, BB Intraperitoneal injection

camptothecin, 6 mg/kg, mm Intra-tumoral Implant BST-gel/camp-

tothecin, 24 mg/kg.

Fig. 3. Body weight of mice given camptothecin by different

administrative routes. The control and the BST-gel only group of

mice were sacrificed within 7 days as their tumors reached four times

the initial volume. OO No treatment, Blank BST-gel,m

m

Intra-tumoral implant BST-gel/camptothecin, 24 mg/kg,BB Con-

trol mice no tumor.

Table 1

TGD following camptothecin treatment by intra-tumoral implant or

intra-peritoneal injection

Treatment (TGD)7S.D.

No treatment 6.570.9

BST-gel 6.871.1

BST-gel/camptothecin (0.45 mg) 25.072.7

Camptothecin i.p 6 mg/kg 7.771.3



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topical irritation or any signs of mechanical stress were

observed in the case of solid gel implants.

4. Discussion

We selected camptothecin as a model drug for thisstudy, because its insolubility in water, makes it difficult

to administer systemically by other means and because

of the potential applications of camptothecin and the

insoluble camptothecin analogues in chemotherapy.

Additionally, the pharmacologically important lactone

ring of camptothecin and its analogs is unstable in the

presence of human serum albumin which results in the

conversion of the active drug to the inactive carboxylate

form bound to albumin [1921]. This imposes a severe

pharmacokinetic limitation on the systemic use of

camptothecin and related compounds. An approach to

overcoming this and other shortcomings of camptothe-

cin and its analogs, especially their high systemic toxicity

is to load it into a delivery system such as a chitosan-

based formulations which will protect the drug from

hydrolysis and control its release over a prolonged

period. Since the active drug is dispersed and not

solubilized there is no possibility of chemical reaction

between the active drug and the excipients.

There are three primary mechanisms for the loaded

drug to be released from hydrogels: swelling, diffusion

and degradation. Drug release from chitosan/GP gel

with initial water content of 84% (w/w) occurs through

the diffusion of water through the polymeric matrix and

dissolution of the soluble fraction of the drug. Water istaken up by hydrogels immediately after being exposed

to an aqueous media, the rate of water uptake

depending on the hydrophilicity of the polymer. As

the gel swells the encapsulated drug is released by

diffusion through pores. In a study of the release of

model compounds from chitosan/GP gels [16] it was

found that release occurred largely by diffusion but

could be accelerated by weight loss of the gels. Weight

loss however occurred much more rapidly than drug was

released. This suggested that during the first few hours

there is leaching of excess GP and of water which does

not contribute to the physical crosslinking of the gel.

The physical three-dimensional structure of the gel does

not change with time suggesting there is no substantial

erosion of the polymer matrix. The third mechanism,

which involves degradation of the polymer matrix,

would only occur under in vivo conditions as a result

of enzyme activity.

It is known that chitosans with block structures and

lower degrees of deacetylation (DDAo75%) are more

readily biodegraded due to the presence of blocks of

glucosamine moieties containing acetyl groups that

serve as a substrate for lysozyme[22,23]. In the present

study, we used chitosan (DDA 85%) that has been

shown to degrade in vivo in about 6 months. No effect

of the degree of deacetylation of chitosan on the in vitro

release kinetics was observed but the in vivo release

kinetics would be expected to be different since in this

case the release kinetics is influenced by both the

biodegradation of chitosan and by diffusion.

Camptothecin delivered systemically resulted in aTGD value of 8 days only, presumably due to the short

half-life of the drug and to the fact that the amount that

can be injected is limited by systemic toxicity. Giova-

nella et al. [24] have shown that after intramuscular

injection of camptothecin in Swiss nude mice (NIH high

fertility strain), camptothecin plasma concentrations

decline in a multi-exponential manner, with a mean

terminal elimination half-life of about 10 h. At this rate,

>99.99% of camptothecin is expected to be eliminated

from the systemic circulation within about 3.5 days.

In this study estimation of toxicity of chitosan/GP or

chitosan/GP/CPT was based on changes in body weight

following hydrogel implant. Several published studies

describe the effect of implant of chitosan/GP on the

histology of the surrounding tissue. The effect of

implant in normal tissue has been described by

Molinaro et al.[25]as a mild non-specific inflammatory

reaction. In the present study the hydrogel was

implanted into the tumor. A report of the effect of

chitosan/GP/paclitaxel implanted into the EMT-6

tumor described the histology of the implanted tumors

as showing some degree of necrosis interspersed between

viable tumor tissue with necrosis generally decreasing

away from the center of the tumor[26]. This pattern was

seen for both chitosan/GP implanted tumors and forthose implanted with chitosan/GP/paclitaxel. In the case

of the RIF-1 tumor chitosan/GP without drug appears

to have no tumoricidal effect so we would not expect to

observe the extent of necrosis seen in the EMT6 tumor

following implantation of chitosan/GP. Histological

changes following implant of chitosan/GP or chitosan/

GP/CPT will be investigated in a projected study.

Chitosan has been shown to activate macrophages for

tumoricidal activity in mice and guinea pigs[27]. Again,

since we found no difference in the tumor response

between the untreated mice and those injected intratu-

morally with chitosan/GP it seems likely that chitosan/

GP does not induce this type of tumoricidal activity

against the RIF-1 tumor.

The effectiveness of the polymer implant in delaying

tumor growth clearly demonstrates the importance of

this delivery system in maintaining an inhibitory level of

drug over a long period of time. The main advantages of

the biodegradable polymer implant such as chitosan/GP

used for the delivery of camptothecin to the mouse

tumor are the high intra-tumoral concentrations of drug

attainable, low systemic toxicity and the extended period

of time over which the drug can be released in the

tumor. The dose of camptothecin delivered using the

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implants was 24 mg/kg, which is 3 times the mean lethal

dose for C3H mice, for the implants the delayed release

of the drug and localization in the tumor prevents toxic

systemic levels being reached whereas a similar dose

delivered by bolus injection would be lethal.

5. Conclusion

Local delivery of chemotherapeutic agent by con-

trolled-release polymers is a new strategy with the

potential to maximize the anti-tumor effect of a drug

and reduce systemic toxicity. In this study, we have

demonstrated the effectiveness of using the biodegradable

chitosan polymer to deliver high doses of camptothecin

locally to a mouse tumor model. Growth of tumors

treated in this fashion was retarded for significantly

longer periods than were tumors treated with systemically

administered camptothecin. Camptothecin delivered by

intra-tumoral implant showed no toxicity in terms of

weight loss. The system formulated with camptothecin

was found to be stable and the release profiles of a

formulation with chitosan andb-GP showed almost zero-

order release kinetics in the first four weeks after an initial

burst of less than 5% in the first day.

These findings show chitosan/GP gel to be a safe,

effective, homogeneous, injectable and stable formulation

for delivery of camptothecin and this approach represents

an attractive technology platform for the delivery of

other clinically important hydrophobic drugs such as

taxol and tetracycline. The mechanism of gelation, which

does not involve covalent cross-linkers, organic solvent ordetergents, combined with a controllable residence time,

renders this injectable biomaterial uniquely compatible

with sensitive chemotherapeutic agents.

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