an in vivo evaluation of docetaxel delivered intratumorally in ......hnscc, the response rates with...

ORIGINAL ARTICLE

An In Vivo Evaluation of Docetaxel DeliveredIntratumorally in Head and NeckSquamous Cell CarcinomaGeorge H. Yoo, MD; Geetha Subramanian, MD; Ramesh R. Boinpally, PhD; Andrew Iskander, MD;Nasfat Shehadeh, MD; Jeffrey Oliver, BS; Waleed Ezzat, BS; Marie P. Piechocki, PhD; John F. Ensley, MD;Ho-Sheng Lin, MD; Terry Y. Shibuya, MD; Lisa Polin, PhD; Ralph E. Parchment, PhD

Objective: To identify activity and biological mecha-nisms of intratumoral (IT) docetaxel on head and necksquamous cell carcinoma (HNSCC).

Methods: Docetaxel IT therapy was tested in xenograftmodels of 2 HNSCC lines, HN30 and HN12. The overalland disease-free survival rates, tumor growth, and toxiceffects were measured. The pharmacokinetic profiles ofdocetaxel in plasma and tumor were compared after ITand intravenous (IV) administration. Comparisons be-tween common and supradoses of docetaxel with re-gard to expression of regulators in the cell cycle, apop-tosis, and signal transduction pathways were determinedusing Western blot analysis.

Results: In the HN30 and HN12 xenograft models, ITdocetaxel improved overall as well as disease-free sur-vival and reversed tumor growth. The only toxic effectsnoted were local (alopecia and skin breakdown). Skinbreakdown resolved in all cases. At equivalent dosing lev-els, IT docetaxel achieved a 26-fold higher peak tumorconcentration and 24-fold longer tumor exposure than

IV treatment. Furthermore, limited plasma exposure wasnoted with IT docetaxel. Supradose levels of docetaxelproduced distinct protein expression patterns for regu-lators of the cell cycle (cyclins A and B, p21, and p27),apoptosis (cleaved caspase-3 and cleaved PARP), and sig-nal transduction (EGFR, pEGFR, pc-Jun, and pERK) inHNSCC, which supports a distinctive mechanism of ac-tion for supradose docetaxel levels. Since levels of cleavedcaspase-3 and PARP, markers of apoptosis, were only el-evated with lower doses, the observed cell death at su-pradose levels was probably due to necrosis.

Conclusions: Injections of IT docetaxel at usual and su-pradoses are associated with a pharmacokinetic profileand biological mechanism distinct from those observedwith usual IV doses. It is calculated that IT therapy inmen will increase peak concentrations of docetaxel in tu-mors by 1000-fold over the conventional IV dose usedclinically. These preclinical results support further test-ing of IT docetaxel in HNSCC.

Arch Otolaryngol Head Neck Surg. 2005;131:418-429

T HE TREATMENT FOR AD-vanced head and neck can-cer includes surgery, radio-therapy, chemotherapy, ormore commonly a com-

bined treatment regimen. Platinum-based chemoradiotherapy compared withradiotherapy alone has been shown to im-prove survival for nasopharyngeal carci-noma1 and unresectable2,3 and oropharyn-geal4 squamous cell carcinoma (SCC).When cisplatin was used together withfluorouracil, organ preservation of the lar-ynx5 and hypopharynx6 was achieved.Chemoradiation using cisplatin aftersurgical resection has demonstrated a ben-efit in 5-year overall survival for ad-vanced head and neck cancer (53% vs 40%,P.02).7 Although systemic cisplatin che-motherapy has significantly reduced theincidence of distant metastasis,5,8 local-regional failure frequently occurs. This has

led to the testing of newer chemotherapyagents. Docetaxel is a taxane that has sig-nificant antitumor effects and is cur-rently being tested in patients with headand neck SCC (HNSCC).9-14

Taxanes promote tubulin assembly inmicrotubules, inhibit depolymerization, actas a mitotic spindle poison, and induce amitotic block in proliferating cells.15 Fur-thermore, taxanes induce G2M arrest andp53-independent apoptosis. Taxanes havebeen shown to improve survival in breast,16

lung,17 and ovarian18 cancers. Although in-travenous (IV) taxanes (paclitaxel anddocetaxel) in combination with 1 or moreagents (cisplatin, fluorouracil, and/or leu-covorin) have demonstrated high clini-cal response rates in newly diagnosedHNSCC (75%-100%),10,12,14,19 in recur-rent disease the response rates are not ashigh (13%-77%); most range between 20%and 40%.20,21 In recurrent and pretreated

Author Affiliations:Departments ofOtolaryngology–Head and NeckSurgery (Drs Yoo, Subramanian,Iskander, Piechocki, Ensley, andLin and Messrs Oliver andEzzat) and Medicine, Divisionof Hematology/Oncology(Drs Boinpally, Shehadeh,Ensley, Polin, and Parchment),Wayne State University andBarbara Ann Karmanos CancerInstitute, Detroit, Mich; andDepartment ofOtolaryngology–Head & NeckSurgery (Dr Shibuya),University of California IrvineCollege of Medicine, Irvine.Financial Disclosure: None.

(REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 131, MAY 2005 WWW.ARCHOTO.COM418

©2005 American Medical Association. All rights reserved.

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HNSCC, the response rates with docetaxel in combina-tion with other agents are only 16% to 24%.11,22 Phase 3trials are ongoing to determine the added survival ben-efit of taxanes. A phase 3 trial in metastatic and recur-rent HNSCC comparing (1) fluorouracil plus cisplatinvs (2) taxol plus cisplatin did not demonstrate an im-provement in survival with taxol plus cisplatin.23 Phase3 trials using taxane combinations are ongoing in newlydiagnosed HNSCC as well.

Tumor cell death induced by a chemotherapeutic agentis proportional to drug concentration and time of expo-sure. Higher tumor concentrations and longer exposureswill have a greater cytotoxic effect. Drug resistance can de-velop due to low drug delivery into tumor cells using tra-ditional IV chemotherapy.24 Intratumoral (IT) chemo-therapy focuses high drug concentrations in solid tumorsand limits total body exposure to the cytotoxic agent re-sulting in increased dose-related cell killing and reducedsystemic toxic effects. Preclinical in vivo studies have shownantitumor activities using IT chemotherapy, as recently re-viewed.25,26 Most IT trials have used cisplatin or ethanol,whereas IT taxanes have not been studied. We have cho-sen to study docetaxel instead of cisplatin because IT trialswith cisplatin have not demonstrated improvement in over-all survival.27

METHODS

DOCETAXEL, CELL LINES,AND HNSCC XENOGRAFT MODEL

Docetaxel (Taxotere; Aventis Pharmaceuticals, Paris, France) wasdissolved in ethanol at 10 mg/mL and stored at –20°C. Serial di-lutions were prepared to make final desired concentrations. Thefinal ethanol concentration was less than 0.05% by volume. TheHNSCC cell lines (HN6, HN12, and HN30) were grown in Dul-becco Modified Eagle Medium with 10% fetal calf serum. Thep53 gene is mutated in HN6 and HN12, while HN30 has a wild-type p53 gene.28 All mouse studies were conducted in compli-ance with and approval from Wayne State University Institu-tional Animal Care and Use Committee–Division of LaboratoryAnimal Research. Female CB17 SCID mice (severe combined im-munodeficiency disorder), aged 6 to 11 weeks, were obtained fromHarlan (Frederick, Md). Cells (15�106) were washed twice inphosphate-buffered saline and suspended in 0.1 mL of phosphate-buffered saline and 0.1 mL of Matrigel (Becton Dickinson, Bed-ford, Mass) for a total volume of 0.2 mL per injection per site.One flank of each mouse was injected with the same cell line. Tu-mor nodule growth was monitored by weekly measurements.

INTRATUMORAL THERAPY IN HNSCCXENOGRAFT SCID MODEL

After tumor nodules formed (maximum diameter, 7-10 mm),the mice were randomized before tumor cell injection, and ITdocetaxel injections were performed along with control injec-tions (diluent alone). Two docetaxel dose groups (low-dose[LD], 7.5 mg/kg per injection; high-dose [HD], 15 mg/kg perinjection) were IT injected twice a week for 6 weeks. The totalweekly IT doses were within the range of commonly used IVdoses in mice (20-40 mg/kg infused weekly).29 An IT therapycourse consisted of 2 IT injections of docetaxel (1 each on days1 and 3) every week for 2 consecutive 3-week cycles. Follow-ing an IT therapy course, mice were observed for 6 more weeks

until day 85 if a complete or partial regression of tumor wasobserved or no change in tumor size was found. At day 95, allsurviving mice were humanely killed. At any time point whenno tumor was noted, IT therapy was discontinued and mice wereobserved until day 95. Weight, tumor size, survival, and toxiceffects were monitored throughout the course of this study.

ANTITUMOR ACTIVITY IN EXPERIMENTALVS CONTROL GROUPS

During and after IT therapy, tumor size and overall and disease-free survival rates were determined for experimental groups andcompared with controls. Tumor nodules were weighed di-rectly at the time mice were killed; otherwise, tumor weightswere estimated using the following formula: length�width insquare centimeters�0.5�1 g/cm3.

Tumor length and width were measured by calipers. Tumorweight was not directly measured; rather, size (volume) was usedto estimate tumor weight. The density of tissue was assumed tobe 1 g/cm3. In human trials, tumor response criteria are often de-termined by using the sum of the largest bidimensional measure-ments, which is considered standard. Therefore, human trials (bi-dimensional) and mouse experiments (length and width) aresimilar in the use of 2 measurements. Given that the same labo-ratory assistant performed the IT docetaxel injections and mea-sured the tumor size, the observer was not blinded to the treat-ment arm. Tumor growth inhibition was estimated also at the timethe mice were killed by using a treatment-to-control (T/C) value(control group was injected with IT diluent): T/C value (percent-age)=median tumor weight in the treatment group/median tu-mor weight in the control group�100. If the T/C value was greaterthan 42%, the treatment was considered not active; 10% to 42%,minimally active; and less than 10%, highly active.

CLINICAL ASSESSMENTS FOLLOWING ITDOCETAXEL INJECTIONS

Tumors were measured 3 times a week by calipers in experi-mental and control groups. Mice were evaluated for weight loss,scruffy appearance, listlessness, uncoordinated movements, in-ability to grasp a pencil, and walking splay footed. All dead miceunderwent necropsy to evaluate spleen size, evidence of gas-trointestinal toxic effects, and the condition of the liver and kid-neys. Mice were killed by carbon dioxide narcosis and cervicaldislocation if (1) tumors reached 1500 mg or 10% of the ani-mal’s body weight, (2) any open lesion appeared, or (3) weightloss reached more than 20% of body weight.

PILOT PK STUDY

Because the pharmacokinetic (PK) profile of IT docetaxel wasunknown, a pilot study was performed in the HN12 xenograftmodel to obtain data to plan the full-scale study. Sampling pointswere chosen in 2 groups of 6 mice at 6, 12, 24, 48, 96, and 192hours after a single IT injection.

FULL-SCALE IV AND IT PK STUDY

The sampling points were chosen based on the PK data from aprevious pilot study conducted in 2 groups of 6 mice each at 6time points. For each treatment (IT and IV), 45 mice bearingHN12 tumors were divided at random into 15 groups of 3. Onegroup served as a control and received no treatment. Each ani-mal in the remaining 14 groups was injected with either IV orIT docetaxel (15 mg/kg). For both the IV and IT study, a totalof 90 mice were used. The animals were anesthetized for 2 min-utes in carbon dioxide starting 2 minutes prior to each sched-




uled time before injection (no treatment group), at injection,5, 15, and 30 minutes after injection, and 1, 2, 3, 6, 10, 24, 32,48, 72, 96, and 192 hours after injection. Blood was collected,via intracardiac puncture, in a 1-mL tuberculin syringe pre-loaded with 0.1 mL of heparin sodium, microfuged for 5 min-utes at 14 000g at room temperature, and the plasma fractionwas separated, weighed, and immediately frozen. The dilutionfactor due to heparin was calculated from the total volume ofheparinized plasma recovered. Unconscious animals were killedby cervical dislocation, and tumor was harvested in individualpreweighed tubes and quick-frozen in a dry-ice bath. All thesamples were stored frozen at –80°C until assay.

DOCETAXEL ASSAY

Docetaxel in the biological samples was quantified using the modi-fied, reversed-phase, high-performance liquid chromatography(HPLC) method of Lee et al30 with paclitaxel as internal stan-

dard. A known volume of frozen heparinized plasma (weighedat the time of storage) was spiked with 10 µL of paclitaxel solu-tion (100 µg/mL) as internal standard and thawed under 3 mLof acetonitrile mixed thoroughly by vortex. After centrifugationat 4°C and 4500 rpm for 10 minutes, the supernatant was trans-ferred to another glass tube and evaporated at approximately 45°Cunder a gentle stream of nitrogen. The residue was reconsti-tuted in 150 µL of mobile phase, and 100 µL were injected intothe HPLC system. Concentrations were corrected for the dilu-tion with 0.1 mL of heparin used as anticoagulant; any samplesdiluted greater than 10-fold were excluded from analysis.

For tumor analysis, specimens were dried of surface mois-ture, accurately weighed, and homogenized in 3 mL of acetoni-trile after adding the internal standard. A Polytron PT 2100 ho-mogenizer (Glenmills, Clifton, NJ) fitted with a Polytron PT-DA2112/2EC probe was used. The homogenate was centrifuged, andthe acetonitrile extract was removed and evaporated as with theplasma samples. The residue was redissolved in 150 µL of mo-bile phase and 100 µL were injected into the HPLC system.

HPLC CONDITIONS

A Waters Nova-Pak C18 (Waters Corp, Milford, Mass) 4-µm(3.9�300 mm) column maintained at 25°C was used in the analy-sis. The mobile phase was 50% acetonitrile in 0.1% phosphoricacid in water at a flow rate of 1.0 mL/min, and the analytes weredetected at 200 nm. Tissue homogenates and plasma from un-treated (control) animals were analyzed to prove lack of inter-fering peaks. Some of these as well as some drug-containingsamples were spiked with standards after initial analysis to con-firm the analyte peaks by coelution with standards. The reten-tion times for docetaxel and paclitaxel were 6.9 and 7.9 min-utes, respectively. Linearity was obtained in the range of 0.1 to100 µg/mL of docetaxel (r2�0.99) with an intraday/interday co-efficient of variation lower than 9%. There were no differencesin standard curve peak areas of standards from neat samples vsspiked tissue homogenates from untreated animals, which in-dicates complete analyte recovery from the homogenates.

PK ANALYSIS

The measured plasma or tumor concentration–time data weresubjected to PK modeling using noncompartmental methods(WinNonlin 4.0.1 software; Pharsight Corporation, MountainView, Calif ). Values for half-life, apparent volume of distribu-tion, drug clearance, and area under the concentration-time curve(AUC) were calculated for the single doses administered by theIT and IV routes.

WESTERN BLOT ANALYSIS

Cells (1.2�106) were plated (100 m2) in 6 mL of Dulbecco Modi-fied Eagle Medium with 10% fetal calf serum. After 48 hours of

Table 1. Overall and Disease-Free 95-Day Survival in Intratumoral Docetaxel and Control Groupsof HN30 and HN12 HNSCC Xenograft Mice

Dose Group

HN30 HN12

Overall, % Disease-Free, % Overall, % Disease-Free, %

Diluent (control) 0 0 0 07.5 mg/kg per injection 100 83 NA NA15 mg/kg per injection 100 100 100 100

Abbreviations: HNSCC, head and neck squamous cell carcinoma; NA, dose not administered to the HN12 group.

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7.5 mg/kg per Injection15 mg/kg per Injection

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1 8 15 22 29 36 43 50 57 64 71 78 85

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15 mg/kg per Injection

Figure 1. Changes in tumor weight in mice following intratumoral delivery ofdocetaxel or control (diluent) in (A) HN30 and (B) HN12 xenograft head andneck squamous cell carcinoma models.




incubation with the viruses, the cells were washed with phosphate-buffered saline. Total cell lysates were prepared by sonicating andincubating the cells in RIPA buffer (150mM sodium chloride, 1%Triton X-100, 1% sodium deoxycholate, 0.4% sodium dodecylsulfate, 20mM EDTA, and 50mM Tris, pH 7.4) for 1 hour at 4°C.Equal amounts of protein from each sample were subjected to7% to 14% sodium dodecyl sulfate–polyacrylamide gel electro-phoresis and transferred to a nitrocellulose membrane (BioRad,Hercules, Calif ). The membrane was blocked with Blotto-Tween (5% nonfat milk, 0.05% Tween 20, 0.9% sodium chlo-ride, and 50mM Tris, pH 7.5) and incubated with the primaryantibodies. Primary antibodies were obtained from NeoMark-ers, Fremont, Calif (p53 and p27 [Kip1]); Santa Cruz Biotech-nology Inc, Santa Cruz, Calif (p21, cyclin A, Skp 2 [p45], andp63Ser-c-Jun); BD PharMingen, San Diego, Calif (cyclin B1); CellSignaling Technology Inc, Beverly, Mass (Bax, caspase-3, andcleaved caspase-3); and Zymed, South San Francisco, Calif (PARPandcleavedPARP).Asecondaryantibody,horseradishperoxidase–conjugated IgG was incubated with membranes and developedaccording to enhanced chemiluminescence protocol using Su-perSignal West Pico (Pierce Biotechnology, Rockford, Ill).

RESULTS

SURVIVAL AND TUMOR RESPONSEUSING IT DOCETAXEL IN HN30 ANDHN12 HNSCC XENOGRAFT MODELS

In the HN30 xenograft model, the overall survival at 95days in the HD (15 mg/kg per injection), LD (7.5 mg/kgper injection), and control (diluent only) groups were100%, 100%, and 0%, respectively (Table1). The disease-free survival at 95 days for the HD, LD, and control groupswere 100%, 83%, and 0%, respectively. The growth ofHN30 xenografts was decreased between HD vs controland LD vs control (Figure 1A). The T/C ratios were 0%for both the HD and LD groups; therefore, IT therapy atHD and LD demonstrated highly active T/C values(�10%). Since HD therapy was associated with an im-proved disease-free survival and was well tolerated in the

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Figure 2. Changes in body weight of SCID mice (severe combined immunodeficiency disorder) treated with intratumoral docetaxel or control (diluent) in (A) HN30and (B) HN12 xenograft head and neck squamous cell carcinoma models.

Table 2. Local Toxic Effects in Intratumoral Docetaxel and Control Groups of HN30 and HN12 HNSCC Xenograft Mice*

Dose Group

HN30 HN12

Total Alopecia Skin Erosion Total Alopecia Skin Erosion

Diluent (control) 6 0 (0) 1 (17) 7 0 (0) 6 (86)7.5 mg/kg per injection 6 5 (83) 2 (33) NA NA NA15 mg/kg per injection 6 6 (100) 5 (83) 7 6 (88) 7 (100)

Abbreviations: HNSCC, head and neck squamous cell carcinoma; NA, dose not administered to the HN12 group.*Unless otherwise indicated, data are reported as number (percentage) of mice.




HN30 xenograft model, the HD was thought to be therecommended IT dose in the HN12 model and for phar-macokinetic studies. In the HN12 HNSCC xenograftmodel, the overall survival at 95 days for HD and con-trol groups were 100% and 0%, respectively (Table 1).The disease-free survival at 95 days for treated and con-trol groups were 100% and 0%, respectively. The growthof HN12 xenografts was significantly decreased in HDvs control groups (Figure 1B). Intratumoral therapy dem-onstrated a highly active T/C value (�10%).

TOXIC EFFECTS OF DOCETAXELBY IT INJECTION IN SCID AND SUBCUTANEOUS

INJECTION IN BALB/c MICE

Throughout the study, no SCID mice displayed a scruffyappearance, listlessness, uncoordinated movements, in-ability to grasp a pencil, or splay-footed walking. Weightloss, used as a broad measure of systemic toxic effects,was not significantly different between experimental andcontrol groups (Figure 2). A noticeable difference wasfound in local toxic effects (skin breakdown and alope-cia) between LD and HD groups compared with controlgroups (Table 2). Since alopecia and skin erosions canbe caused by tumor progression or necrosis, subcutane-ous injections were performed in BALB/c mice withouttumors on the same schedule and with the same dosegroups as IT injections. No significant weight loss(Figure 3A) was observed in the groups of mice in-jected with 7.5 or 15 mg/kg per injection (days 1 and 3every week for 2 consecutive 3-week cycles). Although

both doses caused alopecia, the alopecia resolved in moremice in the LD group than in the HD group (Figure 3B).At both doses, skin erosion occurred in 100% of mice;however, these erosions resolved more slowly in the HDgroup than in the LD group (Figure 3C). Therefore, thelocal toxic effects observed with IT and subcutaneous in-jections of docetaxel appear to be related to agent andnot to tumor progression or necrosis.

PK PROFILE OF IT AND IV DOCETAXEL

Although plasma PKs of IV docetaxel in mice have beendescribed,15 tumor PKs after IT injections have not. There-fore, a pilot study was performed to define appropriate PKsampling time points for a definitive PK study. In the pilotstudy, the PKs of docetaxel after IT delivery were com-pared with those after IV delivery using a single injectionof docetaxel (15 mg/kg) followed by sampling at 6, 12, 24,48, 96, and 192 hours (1 mouse per time point) in the HN12HNSCC xenograft mouse model. The time course of plasmaand tumor concentrations are shown inFigure4A. Plasmadocetaxel levels were undetectable for both IT and IV de-livery even at the initial 6-hour evaluation. Since the tu-mor docetaxel levels were significantly higher in IT thanin IV samples at early time points, a definitive PK studywas designed to compare tumor docetaxel exposure fol-lowing IV and IT therapy and quantify the gain in tumordrug exposure due to IT administration.

The average tumor weights for IV (257 mg; range, 87-503 mg) and IT mice (228 mg; range, 85-318 mg) weresimilar. Tumor and plasma were sampled at 5, 15, 30,

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Figure 3. Toxic effects on BALB/c mice of subcutaneously injected docetaxel: (A) weight loss; (B) alopecia; and (C) skin erosion.




and 60 minutes and 2, 3, 6, 12, 24, 36, 48, 72, 96, and120 hours. The time course of mean plasma concentra-tions of docetaxel and PK parameters in tumor-bearingmice following single IV and IT administration (15 mg/kg) are shown in Figure 4C. The plasma concentrationsof the drug declined rapidly in a biexponential mannerfollowing IV administration, and the levels were belowthe limit of quantification after 2 hours (Figure 4C). Incontrast, plasma levels of the drug increased with timeup to 15 minutes after IT injection and declined rela-tively slowly, showing a half-life of 1.6 hours (vs 0.6 hoursfor IV injection). The level of docetaxel was quantifi-able in plasma up to 3 hours after IT injection. Al-though IV injection resulted in very high plasma levelsinitially, beginning at 30 minutes after drug administra-tion, plasma levels of docetaxel following IT injection weresustained at slightly higher concentrations, most likelydue to the reservoir effect of tumor tissue. Although theoverall systemic exposure (plasma AUC) and clearancefor docetaxel after IT injection were equivalent to thoseof IV injection, the peak plasma concentration for IT de-livery was just 20% of the maximum concentration of theIV injection.

The mean tumor concentrations of docetaxel and thetumor PK parameters are shown in Figure 4B and de-tailed in Table 3. After IV injection, docetaxel was rap-idly taken up by tumor, and the peak levels were at-tained at 30 minutes. Tumor levels of the drug declinedrapidly in a biexponential fashion following IT injec-tion. The tumor levels of docetaxel always remained higherin the IT samples than in the IV samples, especially withinthe first 24 hours, in some cases 26 times higher(Table 4). Although the terminal elimination half-lives of docetaxel in tumor following IV and IT routes ofadministration were comparable, higher tumor expo-sure of docetaxel was achieved through IT injection. Ra-tios (IT-IV) of AUC and concentration of docetaxel intumor following IV and IT injections as a function of timeare listed in Table 4. Although the overall gain in tumorexposure to the drug following a single IT dose vs a singleIV dose was 2.7-fold, the tumor exposure remained greaterthan 10-fold for up to 10 hours (Table 4). Thus, IT therapywith docetaxel results in supraexposure to the drug lo-cally in the tumor. The time that mean tumor docetaxellevels remained greater than 0.5 or 1.0 µg/g was esti-mated to be substantially longer after IT than IV dosing(Table5). These local pharmacologic goals were achievedunder conditions that resulted in equivalent systemic ex-posure to docetaxel (Table 3).

ACTIVITY AND MODULATIONOF BIOMARKERS IN HNSCC CELLS

BY DOCETAXEL SUPRADOSE LEVELS

Since the in vivo IT docetaxel delivery data demonstratedantitumor activity and substantially higher levels, we testedthe effects of supradoses of docetaxel on HNSCC cells invitro. We performed preliminary experiments in whichHNSCC cell cultures were evaluated for cell structure andadhesion at 6, 12, 24, and 48 hours after exposure to 25,100, 500, and 1000 ng/mL of docetaxel. At 24 hours of1000 ng/mL exposure, fewer than 10% of HN12 and HN30

cells adhered to culture plates, and 100% of cells were swol-len and flat (Figure 5). At docetaxel levels of 100 ng/mL, most cells were adherent, and fewer than 50% of cellswere swollen and flat. Head and neck SCC cells werehealthy and 95% confluent at 0 ng/mL.

To test if different biological mechanisms mediate theeffects of supralevels of docetaxel in HNSCC, we per-formed Western blot analysis at 6 and 24 hours using 0,25, 100, 500, and 1000 ng/mL of docetaxel. The pro-teins tested were based on existing expression data.31,32

Most proteins tested did not demonstrate a differentialexpression pattern between typical docetaxel levels (�100ng/mL) and supralevels (�500 ng/ml) except for cer-tain regulators of the cell cycle (cyclins A and B, p21, andp27) (Figure6), apoptosis (cleaved caspase-3 and cleavedPARP) (Figure 7), and signal transduction (EGFR,pEGFR, pc-Jun, and pERK) (Figure 8).

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Figure 4. Changes in docetaxel levels in mice following intratumoral (IT) andintravenous (IV) administration of a single injection (15 mg/kg) in the pilot(A) and definitive (B and C) pharmacokinetic studies of HN12 xenografts.




In general, the expression pattern of cyclin A, cyclinB, p21, and p27 proteins in response to docetaxel wasnot consistent across the 3 HNSCC cell lines (Figure 6).In most test conditions, the expression of these proteinsdid not change significantly across concentrations (25-1000 ng/mL). One notable exception occurred in HN30cells at 24 hours when the expression of cyclin A was de-creased; but at 6 hours, its expression was higher, withincreasing concentration in HN6 cells. For cyclin B, theexpression increased with docetaxel concentration at 6hours, especially in HN6, in a concentration-dependentmanner. At 24 hours, only HN30 demonstrated alteredexpression of cyclin B, which peaked at 100 ng/mL. Theexpression of p21 decreased with increasing concentra-tions in HN12 (6 and 24 hours), HN30 (24 hours), andHN6 cells (24 hours; peaked at 25 ng/mL). Otherwise,p21 expression was higher with increasing concentra-tion at 6 hours for HN6. The expression of p27 in HN12,HN30, and HN6 cells decreased between 25 and 1000ng/mL docetaxel concentrations at 24 hours.

In the apoptosis pathway, caspase-3 and PARP ex-pression along with their respective cleaved products dem-onstrated a consistent pattern of expression in all HNSCCcell lines (Figure 7) and did not change at 6 hours from

500 to 1000 ng/mL concentration of docetaxel vs the con-trol (0 ng/mL). However, at 24 hours, the expression ofcleaved caspase-3 and cleaved PARP peaked at 100 ng/mLand then subsequently decreased with increasing doce-taxel concentration. Furthermore, the level of PARP andcaspase-3 decreased in all HNSCC cell lines at 24 hours.Since cleaved caspase-3 and PARP are markers of apop-tosis, the observed cell death was probably due to ne-crosis. These data suggest that a different mechanism ofaction for supradose levels (1000 ng/mL) is evident byexpression of apoptotic regulators.

Similar to cell cycle regulators, the expression patternof signal transduction was not consistent between HNSCC

Table 3. Plasma and Tumor Pharmacokinetic Data for Docetaxel After a Single IT or IV Injectionin the HN12 Xenograft Mouse Model

Parameter

Plasma Tumor

IV IT IT/IV IV IT IT/IV

Maximum concentration, µg/mL 6.8 1.5 0.2 5.1 132.0 26.0Half-life, h 0.6 1.6 2.7 45.7 50.6 1.1AUC0-t, µg � h/mL 3.2 2.7 0.8 231.0 625.0 2.7AUC0-�, µg � h/mL 3.5 3.7 1.1 244.0 649.0 2.7Clearance, mL/h per kg 4333.0 4014.0 0.9 61.6 23.1 0.4

Abbreviations: AUC, area under the curve; IT, intratumoral injection; IV, intravenous injection.

Table 4. Single Intratumoral–Single Intravenous Docetaxel Injection Ratios for Tumor Area Under the Curveand Mean Tumor Concentration in the HN12 Xenograft Model

Parameter

Time After Injection, h

0.08 0.25 0.5 1 2 3 6 10 24 32 48 72 96 192

Area under the curve 20 21 23 24 20 16 13 10 5.8 4.9 4.0 3.4 3.1 2.7Mean tumor concentration 20 23 26 22 11 7.2 8.0 1.8 1.1 1.3 1.2 1.5 1.3 1.9

Table 5. Estimated Period of Docetaxel ConcentrationsGreater Than 0.5 and 1.0 µg/mL in the HN12 XenograftMouse Model Following a Single Intravenousor Intratumoral Dose

Tumor DocetaxelConcentration, µg/g

Time Greater ThanConcentration, h

Intravenous Intratumoral

0.5 130 1601 85 110

100 ng/mL0 ng/mL 1000 ng/mL

100 ng/mL0 ng/mL 1000 ng/mL

HN12 Cells

HN30 Cells

Figure 5. Microscopic views of HN12 and HN30 cells after 24 hours ofexposure to docetaxel at 0, 100, and 1000 ng/mL.




cell lines (Figure 8). Expression of EGFR decreased withdocetaxel concentration and was concentration depen-dent at 24 hours for HN12 and HN30 cell lines. Other-wise, HN6 (6 and 24 hours), HN30 (6 hours), and HN12(6 hours) cell lines showed no change in expression at anyof the concentrations or time periods tested. The expres-sion of pEGFR increased with increasing concentration at6 hours for HN12. No EGFR expression was detected forHN30 (6 and 24 hours) or HN12 (24 hours). Expressionof pEGFR decreased with increasing concentration at 24hours in the HN6 cell line. The expression of pc-Jun inHN12 cells peaked at 25 ng/mL of docetaxel concentra-tion and then decreased with increasing concentrations at6 and 24 hours. In HN30 and HN6 cells, expression of pc-Jun increased at 6 and 24 hours independent of the doce-taxel concentration. At 6 hours, expression of pERK in-creased slightly with increasing docetaxel concentrationsin HN12 and HN6 cells. In HN30 (6 hours) and HN6 cells(24 hours), pERK expression decreased with increasingdocetaxel concentrations. For HN12 and HN30 cells, the

expression of pERK peaked at 100 ng/mL of docetaxel at24 hours and subsequently decreased at higher concentra-tions (500-1000 ng/mL).

COMMENT

Chemotherapy-induced cytotoxic effects on tumor cellsare proportional to drug concentration and time of ex-posure. Therefore, a higher deliverable IT concentra-tion will have a greater cytotoxic effect. Using tradi-tional IV chemotherapy, drug resistance can result fromlow drug delivery into tumor cells.24 Furthermore, widedistribution throughout the body results in a broad rangeof serious adverse effects. Intratumoral chemotherapy fo-cuses high drug concentrations in solid tumors and lim-its total body exposure to the cytotoxic agent, resultingin increased dose-related killing of tumor cells at re-duced, or at least equivalent, systemic toxic effects. Inthe present HNSCC xenograft mouse study, overall and

HN12

0 25 100 500 1000 0 25 100 500 1000

DocetaxelConcentration,

ng/mL =

HN30

HN6

Cyclin A

HN12

HN30

HN6

Cyclin B

HN12

HN30

HN6

p21

HN12

HN30

HN6

p27

6 Hours 24 Hours

Figure 6. Protein expression patterns of cell cycle regulators in head and neck squamous cell carcinoma cells after exposure to different concentrations ofdocetaxel for 6 and 24 hours.




disease-free survival rates were improved by IT injec-tion of docetaxel. A favorable PK profile of IT deliverywas observed when compared with IV tail vein injec-tions (Tables 3, 4, and 5).

The total weekly IT doses used in this study (7.5 or 15mg/kg per injection twice weekly) were chosen to matchpreviously reported weekly IV doses in mice.29 The maxi-mum concentrations of docetaxel delivered by IT admin-istration were much greater than those from IV injection(132.0 vs 5.1 µg/mL; IT-IV ratio, 26). Earlier peak tumorlevels were found to be 4 µg/mL after IV infusion of doce-taxel (111 mg/m2) in mice with tumors.29 With IT deliv-ery, tumor levels (1.9- to 26-fold) and tumor AUCs (2.7-to 24-fold) remained high throughout the study (�192hours). Since the clearance of docetaxel was also slowerwith IT delivery than with IV (23.1 vs 61.6; ratio, 0.4), timeof tumor exposure to docetaxel was greater with IT injec-tions. The fact that the published IV dose of 111 mg/m2

achieves a peak concentration of 4 µg/mL, whereas the singledose of 45 mg/m2 in the present study achieved 5.1 µg/mL

concentration, suggests that slow tumor penetrationofdoce-taxel from the plasma may limit delivery.

The maximum plasma concentrations were muchlower for IT than for IV delivery (1.5 vs 6.8 µg/mL), andthe plasma half-life of IT injections was longer than thatof IV (1.6 vs 0.6 hours); however, the AUC and clear-ance were equivalent (Table 3). Since clearance of doce-taxel from tumor tissue is slower than from plasma,29 peakplasma levels from IT injections were lower than thosenoted with IV delivery. The lower plasma levels seen withIT injections will decrease the concentration-dependent systemic toxic effects observed with IV de-livery. Since docetaxel undergoes hepatic metabolism andbiliary-fecal excretion and the 2 major metabolites areinactive against tumor cells,29 systemic bioactivation isnot required for docetaxel activity.

After IV infusion of docetaxel in mice with tumors,the peak tumor and plasma levels were 4 and 50 µg/mL,respectively, which means that peak concentration of tu-mor-plasma ratio is approximately 8%.28 Using the rec-

HN12

0 25 100 500 1000 0 25 100 500 1000


ng/mL =

HN30

HN6

Caspase 3

HN12

HN30

HN6

CleavedCaspase 3

HN12

HN30

HN6

PARP

HN12

HN30

HN6

CleavedPARP

6 Hours 24 Hours

Figure 7. Protein expression patterns of regulators of apoptosis in head and neck squamous cell carcinoma cells after exposure to different concentrations ofdocetaxel for 6 and 24 hours.




ommended IV bolus of docetaxel (100 mg/m2) inhumans, the peak plasma concentration was 3.2 µg/mLat 1 hour and 1 ng/mL at 60 hours (AUC=4.81 µg•hr/mL).33 Using this 8% ratio in humans (100 mg/m2),the estimated concentration in the tumor would peak at261 ng/mL, and at 60 hours the concentration would beonly 0.08 ng/mL. Using IT chemotherapy, the entire doseof agent can be delivered into the tumor by intralesionalinjections, and low tumor penetration through IV routescan be overcome. Tumor levels using an IT delivery (100mg/m2; body surface area, 1.78 m2) would be approxi-mately 1000-fold higher than previously reported IV de-livery.ThePKdata, tumor-plasmaratios, collected in thesepreclinicalmodelswill guide the initialdoseand frequencyof administration in future phase 1 and 2 trials.

The toxic effects observed in the present HNSCC xe-nograft models were local (alopecia and skin erosion),not systemic. Higher tumor levels (up to 26-fold) andlower peak plasma concentrations (20%) with IT thanwith IV injection support a relationship between the type

of toxic effect (local vs systemic) and the delivery ap-proach (IT vs IV). Previous reports have demonstratedno significant systematic toxic effects using intradermalinjections of docetaxel (0.9% sodium chloride, polysor-bate 80) in rabbits at doses 3 times those used in hu-mans.29 The systemic dose-limiting toxic effect (neutro-penia) and others (nausea, mucositis, asthenia, diarrhea,peripheral neuropathy, infection, and sodium-wasting ne-phropathy) may be limited with an IT approach.

Most IT trials have used cisplatin or ethanol, whereasIT taxanes have not been studied (Table 6). We havechosen to study docetaxel instead of cisplatin because ITtrials with single-agent cisplatin have not demonstratedimprovement in overall survival.27 The results of 2 double-blind, placebo-controlled, randomized studies of IT cis-platin/epinephrine/gel in patients with recurrent HNSCCwere reported.21 The researchers randomized 179 pa-tients in a 2:1 fashion to receive treatment with eithercisplatin/epinephrine/gel (n=119) or placebo (n=60). Theoverall rate of response lasting 28 days or longer was 29%

HN12

0 25 100 500 1000 0 25 100 500 1000


ng/mL =

HN30

HN6

EGFR

HN12

HN30

HN6

pEGFR

HN12

HN30

HN6

pc-Jun

HN12

HN30

HN6

pERK

6 Hours 24 Hours

Figure 8. Protein expression patterns of signal transduction molecules in head and neck squamous cell carcinoma cells after exposure to different concentrationsof docetaxel for 6 and 24 hours.




in the treatment arm (19% complete response, 10% par-tial response) and 2% in the placebo arm (P�.001). Painduring the injection was the most common adverse event:24% of patients in the treatment arm and 17% in the pla-cebo arm experienced pain within 15 to 20 minutes. Lo-cal toxic effects, including inflammation, bleeding, ero-sion, ulceration, necrosis, and eschar, occurred in 87.4%of patients in the treatment arm and 62.7% in the pla-cebo arm. Other adverse effects included wound infec-tion in 5.9% vs 1.7% and fistula formation in 3.3% vs 0%for patients in the treatment arm vs placebo arm, respec-tively. Four cases of treatment-related cerebrovascularevents (3 in the treatment arm and 1 in the placebo arm)were observed. After implementation of an amendmentto exclude patients with tumors that directly involved orthreatened to invade the carotid artery, no further cere-brovascular events related to treatment were reported.In clinical trials using IT docetaxel, we also expect nosignificant systemic toxic effects (thanks to lower plasmaconcentrations) and no cerebrovascular events (be-cause no epinephrine will be involved).

In the present study, docetaxel’s mechanisms of ac-tion at supradose levels with respect to cell cycle regu-lation, apoptosis, and signal transduction were exam-ined. In a future study, we are planning a detailedhistologic and protein expression analysis using immu-nohistochemical analysis, since only in vitro studies wereperformed in this pilot study. The docetaxel-induced al-tered protein expression pattern of cell cycle regulators(cyclin A, cyclin B, p21, and p27) and signal transduc-tion molecules (EGFR, pEGFR, pc-Jun, and pERK) was

not consistent between HNSCC cell lines. However, theexpression of cleaved caspase-3 and cleaved PARPpeaked at 100 ng/mL and then subsequently decreasedwith increasing concentration at 24 hours in all HNSCCcell lines. This peak of apoptosis at 100 ng/mL did notcorrelate with extensive cell death (cellular swellingand lack of adherence) observed at 500 and 1000 ng/mL. Therefore, cellular death at supradosing levels ap-peared to be predominantly due to necrosis and notapoptosis.

Cell death can occur through apoptosis and/or necro-sis. Apoptosis and necrosis are separate entities with over-lapping mechanisms and probably represent a con-tinuum of cell death mechanisms (Table7).42,43 Apoptosisand necrosis can occur in the same cellular population.Apoptosis cells first will shrink and the nuclei becomecondensed. Necrosis has distinct morphologic features(cellular swelling) that are accompanied by rapid per-meabilization of the plasma membrane and lyses. Ne-crosis usually occurs secondary to physiologic and patho-physiologic stimuli such as infection, ischemia, and injury.The mechanism of chemotherapy has been focused onenhancing apoptosis. Optimizing necrosis (eg, throughtreatment with IT docetaxel) is another pathway to achievetumor cell death.

In conclusion, an antitumor effect independent ofapoptosis pathways and a distinct pharmacokinetic pro-file (supradosing) are attainable with IT delivery of doce-taxel. The identification of altered gene expression in-duced by docetaxel at supradose levels demonstratesdifferent mechanisms, possibly necrosis, in HNSCC cells.The initiation of a phase 1/2 trial using IT docetaxel inpatients with HNSCC is justified by these data.

Submitted for Publication: December 8, 2004; ac-cepted February 2, 2005.Correspondence: George H. Yoo, MD, Department of Oto-laryngology–Head and Neck Surgery, Wayne State Uni-versity, 5E University Health Center, 4201 St Antoine,Detroit, MI 48201.Funding/Support: This work was supported by grants R21DE014878 from the National Institutes of Health/National Institute of Dental and Craniofacial Research;GIA 10161 from Aventis Pharmaceuticals; and P30-

Table 6. Intratumoral Chemotherapy Trialsfor Solid Tumors, Excluding Trials for Skin Cancer,Immunotherapy, and Gene Therapy

Agent,Reference Tumor Type

No. ofPatients Summary

Cisplatin25 Head and neckcancer

82 Doses, 1 to 6 mg/cm3 oftumor; response rate, 50%;complete response, 40%

Cisplatin27 Head and neckcancer

178 Response rate, 29 vs 2%(P = .001); overall survival,no significant difference

Cisplatin34 Liver cancer 6 Pharmacokinetic study; time topeak and half-life longer withIT than IV chemotherapy

Cisplatin35 Solid 4 Tumor regression orstabilization

Cisplatin36 Esophagealcancer

9 Tumor volume reduced in 3patients

P-3237 Solid 17 Response rate, 71%; completeresponse, 41%

Ethanol38 Liver cancer 207 1-, 2-, and 3-y survival, 90%,80%, and 63%, respectively

Ethanol39 Liver cancermetastases

30 Reduction in tumor, n = 20;decreased AFP and CEAlevels



Abbreviations: AFP, alpha-fetoprotein; CEA, carcinoembryonic antigen;IT, intratumor; IV, intravenous.

Table 7. Summary of Apoptosisand Necrosis Characteristics*

Characteristic Apoptosis Necrosis

Cell death Programmed IrreversibleMorphologic

propertiesCell shrinkage, nuclear

chromatincondensation

Cell swelling, membranecollapse

Inflammation None PresentInjury Response to mild injury Response to severe injuryEnergy

dependenceEnergy dependent Energy independent

Pathways Predictable andpredetermined

Independent biochemicalevents

Prevention Possible Difficult to prevent

*Data reported in Kanduc et al42 and Proskuryakov et al.43




CA022453 from the Pharmacology Core of the Meyer L.Prentis Comprehensive Cancer Center.Acknowledgment: We thank Sen-lin Zhou, BS, for tech-nical assistance.

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an in vivo evaluation of docetaxel delivered intratumorally in ......hnscc, the response rates with...

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