in vivo bioavailability and pharmacokinetics of a c-myc antisense

In vivo Bioavailability and Pharmacokinetics of a c-MYCAntisense PhosphorodiamidateMorpholino Oligomer,AVI-4126, in SolidTumorsGayathri R. Devi,1 TomaszM. Beer,2 Christopher L. Corless,2 Vikram Arora,1

Doreen L.Weller,1and Patrick L. Iversen1

Abstract Phosphorodiamidate morpholino oligomers (PMO) inhibit targeted gene expression by prevent-ing ribosomal assembly, thereby preventingmRNA translation. AVI-4126, a PMO targeted againstc-MYC, has been extensively characterized in multiple cancer and other disease models and iscurrently in human clinical trials. A phase I clinical study was conducted to address the issue ofPMObioavailability in malignant tumors surgically excised from patients with adenocarcinoma ofprostate and breast 1day after i.v. administration of a single dose of 90 mg AVI-4126 PMO. Thestudy objectives were to evaluate safety, to determineAVI-4126 concentration in tissue samplesof the tumors, and to examine the distributionof AVI-4126 (margin versus tumor core). Significantconcentrations of intact PMO similar to the animal models were detected in both human prostateand breast tumor tissues with increased distribution in the tumor core for the vascular breasttumors. No serious adverse events (graded according to National Cancer Institute CommonToxi-city Criteria) were reported. Another phase I study was conducted innormal human volunteers toassess AVI-4126 plasmapharmacokinetics following single i.v. administrationof 90mgAVI-4126.Data from both human studies indicated similar plasma concentration-time profile. These studiesshowPMObioavailability in tumor tissue and establish the feasibility of using PMO targeting spe-cific genes inhuman cancer clinical trials.

The c-MYC proto-oncogene, which is frequently overex-pressed in human cancer, plays a critical role in the controlof cell proliferation, differentiation, and apoptosis (1, 2).Several different strategies, including antisense-mediatedinhibition of the gene, have been employed to developnovel drugs that seek to inactivate c-MYC for the treatmentof cancer (2). AVI-4126, a phosphorodiamidate morpholinooligomer (PMO) targeting c-MYC , has an excellent safetyprofile in preclinical animal models and in human clinicaltrials (3). PMOs are neutrally charged antisense agents,wherein the deoxyribose moiety of DNA is replaced witha six-membered morpholine ring and the charged phospho-diester internucleoside linkage is replaced with phosphoro-diamidate linkages (4, 5). PMO antisense molecules are stericblockers and inhibit gene expression by physically preventingbinding or progression of splicing or translational machinerycomponents (5–7). The neutral character of the PMO chem-

istry avoids a variety of potential significant limitations,including the binding of other cellular and extracellularproteins that is observed with charged oligonucleotide chem-istries (8, 9).

The 20-mer AVI-4126 sequence was chosen based onfavorable solubility, efficacy, and potency compared with>100 other antisense c-MYC PMOs targeted to various sitesalong the c-MYC 5V untranslated region, the splice acceptor ofthe first intron, and around the translation start site. Previousstudies have shown that AVI-4126 inhibits c-MYC translationin a sequence-specific manner in cancer cell lines in culture bysteric blockade and missplicing of pre-mRNA, resulting insignificant growth inhibition (10, 11). AVI-4126 has beenobserved to specifically down-regulate c-Myc protein, decreasecyst formation, and improve renal function in a murine modelof polycyctic kidney disease (12); causes growth arrest in arat liver regeneration model (13); and is effective in prevent-ing cardiac restenosis in multiple animal models (14, 15). Thesafety profile of the PMO chemistry, including that of AVI-4126, has been widely studied in animal toxicity models aswell as in human clinical trials after systemic and local routesof administration (3, 16). No serious side effects (graded ac-cording to the National Cancer Institute Common ToxicityCriteria) have been reported. AVI-4126 antisense PMO has alsoshown promising efficacy in tumor models of lung cancer (17)and prostate cancer (3, 18, 19). Therefore, the purpose of thisstudy was to characterize PMO bioavailability in solid tumormodels in animals and in surgically excised malignant tumorsfrom patients after single systemic AVI-4126 administrationand to assess pharmacokinetics and safety.

www.aacrjournals.orgClin Cancer Res 2005;11(10) May15, 2005 3930

Authors’Affiliations: 1AVIBioPharma, Inc., Corvallis, Oregon and 2Oregon Healthand Science University Cancer Institute, Portland, OregonReceived10/13/04; revised 2/16/05; accepted 2/18/05.Grant support: AVIBioPharma, Inc. and Department of Defense grant DAMD17-01-0017 (G.R. Devi).The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Gayathri R. Devi, Comprehensive Cancer Center, DukeUniversity Medical Center, 401MSRB, Box 2606, Durham, NC 27710. Phone:919-668-0410; Fax: 919-681-7970; E-mail: [email protected].

F2005 American Association for Cancer Research.

Cancer Therapy: Preclinical

Research. on January 6, 2019. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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Materials andMethods

OligomersAVI-4126 (c-MYC antisense PMO; 5V-ACGTTGAGGGGCATCGTCGC-

3V) and control scrambled (5V-ACTGTGAGGGCGATCGCTGC-3V) mor-pholino oligomers were synthesized at AVI BioPharma, Inc. (Corvallis,OR) as described previously (4, 18). Purity was >95% as determined byreverse-phase high-performance liquid chromatography (HPLC) andmatrix-assisted laser desorption ionization time of flight massspectroscopy.

Plasmid-based test system for screeningphosphorodiamidate morpholino oligomer antisenseactivityA fusion c-MYC luciferase construct (pCiNeo-myc -LucDA) was

generated in the pCiNeo expression vector (Promega, Madison, WI)as described previously (20). This plasmid features a T7 promoter capableof generating in vitro transcribed RNA from a cloned insert for use in cell-free rabbit reticulocyte in vitro translation reactions and a cytomegalo-virus promoter for constitutive expression in mammalian cells. In vitrotranscription was carried out with T7 Mega script (Ambion, Austin, TX).

Cell-free luciferase assayIn vitro translation was done by mixing rabbit reticulocyte lysate with

known amounts of antisense, scrambled PMOs or vehicle (water)followed by addition of a known amount of the c-MYC/Luc RNA (f1nmol/L final concentration). The Promega luciferase assay reagentprotocol was followed. The percent inhibition of luciferase activitycompared with control was calculated based on readings from aluminometer (Cardinal, Santa Fe, NM).

Cell culturePC-3, LNCaP, DU145, human prostate cancer cell lines, and LLC1

murine Lewis lung cancer cell line were obtained from American TypeCulture Collection (Rockville, MD). The prostate cancer cells werecultured in RPMI 1640 (Hyclone Laboratories, Logan, UT) supple-mented with 10% fetal bovine serum (Life Technologies, Gaithersburg,MD), 100 units/mL penicillin, and 75 units/mL streptomycin at 37jCunder 5% CO2. LLC1 were maintained in DMEF-12 medium (HycloneLaboratories) supplemented with 10% fetal bovine serum, 100 units/mLpenicillin, 100 Ag/mL streptomycin, and 0.25 Ag/mL amphotericin.

AnimalsMale athymic (Ncr nu/nu) nude mice and C57BL/6 mice f4 weeks

old on arrival were obtained from Simonson Laboratories (Gilroy, CA)and were housed in sterile plastic cages at the Laboratory AnimalResources Facility at Oregon State University (Corvallis, OR). Theathymic mice were housed in laminar airflow cabinets in microisolatorcages under pathogen-free conditions with a 12-hour light/dark scheduleand fed autoclaved standard chow and water ad libitum . LNCaP and PC-3cells (2 � 106) were suspended in 150 AL RPMI 1640 plus 10% fetalbovine serum and 150 AL Matrigel (Becton Dickinson, Palo Alto, CA).DU145 cells (5� 106) were suspended in 150 AL RPMI 1640 and injectedvia a 25-gauge needle into the s.c. space of the flank region of 5- to 6-week-old athymic male mice (3, 18). LLC1 cells (2 � 105) were injecteds.c. into the right rear flank of the C57BL/6 mice (17). All animals wereanesthetized with isofluorane before injection of cells. All animalprotocols conformed to the ethical guidelines of the 1975 Declarationof Helsinki and were approved by the Institutional Animal Care and UseCommittee of Oregon State University.

Mouse treatment protocolsThe experiments were started when the tumors of each type of

xenografts were well established and the tumor volumes were between500 and 700 mm3, and the mice were randomly assigned to differentgroups (n = 4-8). AVI-4126 PMO (300 Ag equivalent to a dose of15 mg/kg) dissolved in saline was given either intratumorally (i.t.) or

i.p. as indicated. After 24 hours, the animals were euthanized andtumors were excised and processed for flow cytometry, photomicrog-raphy, histology, and/or preparation of lysates.

Protein expression in mouse tumorsTumor samples were lysed immediately after the animal was

euthanized. Protein estimation was carried out by Bradford proteinassay kit (Bio-Rad, Hercules, CA). Equal amounts of protein in cell ortissue lysates were subjected to 10% or 12% SDS-PAGE under reducingconditions and probed with anti c-Myc polyclonal antibody asdescribed previously (18).

Photomicrography of mouse tumorsTumor slices were placed in plastic molds filled with embedding

medium for frozen tissue processing (Sakura Finetek, Torrance, CA).The molds were wrapped in aluminum foil to protect from light andfrozen in a �80jC freezer. Cryostat sections (5 Am) were cut at OregonState University Veterinary Diagnostic Laboratory. Slides were air driedin the dark for 30 minutes and coverslip mounted using Fluoromount-G mounting medium (Southern Biotechnology Associates, Birming-ham, AL). The photomicrographs were taken with a Nikon Diaphot 300microscope connected to an Olympus (Melville, NY) Magnafire SP-brand digital camera. The exposure times were kept constant for allfluorescent pictures at 25 seconds.

Flow cytometry of mouse tumor cellsTumor cells from animals injected with fluorescein-labeled AVI-

4126 were harvested by digestion with 0.1% collagenase (type I) and50 mg/mL DNase as described previously (21). Dispersed cells werewashed thrice with PBS and resuspended in fluorescence-activated cellsorting buffer. Flow cytometry was carried out at the Oregon StateUniversity Core Facility using the BD FACSCalibur cytometer (BectonDickinson, Mountain View, CA) and data were analyzed using the FCSexpress software.

High-performance liquid chromatography detection ofphosphorodiamidate morpholino oligomer in tumorand organ tissueTumor tissue, liver, and kidney lysates from AVI-4126-treated

animals were prepared as described above and analyzed for presence

of PMO by HPLC analysis as described (22). Briefly, a 10-AL aliquot

(500 ng) of the internal standard PMO (15-mer whose sequence was

derived from a 5V truncation of AVI-4126) was added to all 250-AL

aliquots of tumor, kidney, and liver lysate (0.2 g/mL) samples.

Methanol (300 AL) was added to each sample and the tubes were

vortexed. The tubes were centrifuged for 10 minutes using a high-speed

centrifuge and supernatants were transferred to new Eppendorf tubes.

The pellet was then washed with 100 AL Tris and the wash buffer was

added to the supernatant. The supernatants were heated in a water bath

at 70jC for 10 minutes. The samples were recentrifuged for 10 minutes

and the supernatants were transferred to new Eppendorf tubes.

Methanol was evaporated using a Speed vac (Savant, Farmingdale,

NY) and the samples were finally transferred to clear shell vials and

lyophilized after the addition of 100 AL deionized water to each vial.

The lyophilized samples were reconstituted using 100-AL aliquots of 5Vfluoresceinated DNA (1.0 absorbance units/mL) whose sequence was

complementary to that of AVI-4126 PMO. A set of AVI-4126 standards

was prepared by spiking the PMO into 250-AL aliquots of blank rat

plasma (10, 25, 50, 100, 250, 500, and 1,000 ng/250 AL plasma) along

with the internal standard. The standards were extracted similarly.The samples were analyzed by injecting onto a Dionex DNA Pac

PA-100 column (4 � 250 mm column, Dionex Corp., Sunnyvale, CA)using a Varian autosampler (AI-200) connected to a Varian HPLCpump (model 9010 inert) equipped with a Varian fluorescencedetector (model 9075). The mobile phases [A: 0.025 mol/L Tris-HCl(pH 8); B: 0.025 mol/L Tris (pH 8)/1.0 mol/L NaCl] were preparedusing HPLC-grade water and reagents and filtered through a 0.2 Am

www.aacrjournals.org Clin Cancer Res 2005;11(10)May15, 20053931

Bioavailability and Pharmacokinetics of AVI-4126



filter before use. The gradient program employed was [90-10% B] at

0 minute and [55-45% B] at 20 minutes while the pump was held at a

flow rate of 1.5 mL/min. The runs were monitored at excitation and

emission wavelengths of 494 and 518 nm, respectively.

Pharmacokinetic studyA phase I single center, open label, and dose escalating study was

conducted to evaluate the safety (3) and pharmacokinetics of AVI-4126

given i.v. in normal healthy male and female subjects. Protocol

specifications and a detailed time and events schedule were prepared by

MDS Harris (Lincoln, NE) with local ethics committee approval. Five

dose levels were evaluated with six normal healthy subjects enrolled in

each cohort after screening medical history, examination, and

laboratory tests had shown no clinically significant abnormalities.

Cohorts 1 (three males and three females), 2 (two males, four females),

3 (three males, three females), 4 (two males, four females), and 5 (four

males and two females) received 1, 3, 10, 30, and 90 mg AVI-4126,

respectively, given as a slow (10-15 seconds) i.v. bolus/push. The

subjects in all five dose levels were in the fed state. They were confined

to the clinic through the 72-hour post-dose events. The pharmacoki-

netic variables were studied for the highest dose (90 mg). Blood draw

was conducted at the following time points: 0.033, 0.083, 0.167, 0.25,

0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hours following AVI-4126

administration.

Pharmacokinetic analysisThe data were evaluated with the assistance of the computer program

PKCALC as published previously (23). The i.v. plasma concentrations

measured were fit to Eq. A (24):

CðtÞ ¼ Ae��t þ Be��t ðAÞ

where C(t) is the plasma concentration at time t , A and B are

intercept terms, a is a distribution rate constant, and h is an

elimination rate constant. All data are expressed as mean F SE as

determined by the computer program InStat 2 (GraphPad, San Diego,

CA). Graphs were prepared with Prism version 3.0 (GraphPad).

Phase I study of AVI-4126 localization in human solidtumorsIn an exploratory analysis, we sought to characterize the localization

of AVI-4126 in human solid tumors.Patients. Adult, good performance status (Eastern Cooperative

Oncology Group PS 0 or 1) patients with cytologically orhistologically confirmed adenocarcinoma of the breast, colon,prostate, or non–small cell lung cancer who had a tumor at least1.0 � 1.0 cm and were scheduled for surgical resection were eligible.Patients could not have metastases, active infection, or another seriousmedical condition; could not be pregnant or nursing; could not havereceived an investigational agent within 30 days or any priorchemotherapy, hormonal therapy, or radiation therapy; and had tohave adequate hematologic, renal, and hepatic function (hemoglobin>11 g/dL for men and >10 g/dL for women; serum creatinine Vinstitutional upper limits of normal; serum bilirubin < upper limits ofnormal; aspartate aminotransferase < 1.5 � upper limits of normal).All patients provided signed informed consent and the study wasapproved by the Institutional Review Board of the Oregon Healthand Science University.

Procedures. Pretreatment evaluation included history and physical

examination, urinalysis, serum pregnancy test, complete blood count

with automated differential, and serum chemistries. AVI-4126 (90 mg)

was given by a slow i.v. push (f10-15 seconds) 16 to 30 hours before

the scheduled surgery. Vital signs were monitored before and

30 minutes after dosing, before hospital discharge, and 7 to 14 days

later. Complete blood count and serum chemistries were obtained

before hospital discharge and again 7 to 14 days later. EDTA-stabilized

plasma was collected for AVI-4126 concentration analysis immediately

before AVI-4126 dosing, 30 minutes later, before anesthesia, and after

anesthesia recovery. Adverse events were recorded 30 minutes after

dosing and again 7 to 14 days later. Tissue specimens that included the

core and margin of the tumor as well as adjacent normal tissue were

collected with the goal of measurements of the concentration and

distribution of AVI-4126.

Results

AVI-4126 activity in a plasmid-based in vitro screening system.To ascertain the sequence specificity of the PMO, AVI-4126, aplasmid-based test system was used for cell-free screening. Afusion construct, pCiNeo-myc-LucDA, was generated by sub-cloning a small segment of the c-MYC gene that includes theAUG translation start site followed by luciferase into thepCiNeo expression vector. In vitro translation was done bymixing the rabbit reticulocyte lysate with known amounts ofantisense, scrambled oligomers or vehicle (water) followed byaddition of a known amount of the c-MYC/Luc in vitrotranscribed RNA. The relative inhibition of luciferase activityin the presence of various concentrations of PMO comparedwith the vehicle control was calculated based on the readingsfrom a luminometer. The data in Fig. 1 reveal a dose-dependent and specific inhibition of luciferase activity, IC50 of100 nmol/L in the presence of increasing concentrations ofthe antisense c-MYC PMO (AVI-4126) compared with thescrambled PMO control.

Differential bioavailability of AVI-4126 in murine solid tumorsmodels. Three different tumor models with varying degrees ofvascularity (LLC1 syngeneic lung tumor, LNCaP prostatexenograft, and PC-3 prostate xenograft) were employed toexplore the relationship between tumor blood supply andaccumulation of AVI-4126 in the tumor. In each case, a singledose (300 Ag/mouse) of AVI-4126 was given to the mice i.p.and/or i.t. The tumors were resected 24 hours after AVI-4126,


Fig. 1. Plasmid-based test system for screening AVI-4126 PMO sequencespecificity and antisense inhibition of c-MYC mRNA in the region from 5Vuntranslated region in to theAUG translation start site with the luciferase reportergene. In vitro translation was done by mixing rabbit reticulocyte lysate with variousconcentrations of vehicle (water), antisense, or scrambled PMOs in the presence ofc-MYC luciferase RNA. Points, mean (n = 3) to reveal the luciferase light units in thelysates normalized for protein content to show specificity of the antisense c-MYCPMO in comparisonwith a 70% identical but scrambled sequence; bars, SE.




scrambled PMO, or saline administration and subjected to flowcytometric analysis, preparation of cryosections, and tumorlysates. Significant AVI-4126 bioavailability was detected up to24 hours after a single i.p. administration in the LLC1 lungsyngeneic tumors (Fig. 2) and LNCaP prostate xenografttumors, both of which are highly vascular (Fig. 3) Immunoblotanalysis of the respective tumor lysates (Figs. 2A and 3A)revealed specific inhibition of target c-Myc protein levels in theAVI-4126 PMO-treated tumors compared with the controlgroups. Quantitation of AVI-4126 PMO levels in the tissuelysates from the tumors was carried out by HPLC analysis. Theelution order of each chromatogram is AVI-4126, the internalstandard, and the excess fluoresceinated DNA probe. The peakcorresponding to full-length AVI-4126 was readily observed inboth LLC1 (Fig. 2B and C) and LNCaP (Fig. 3B and C) tumortissue samples as shown in the representative chromatograms.This analytic technique is capable of resolving peaks resultingfrom (N - 1)-mers and truncated versions of AVI-4126, but

neither was detected in the tissue or tumor lysates. Significantlevels of AVI-4126 PMO were detected, which correlated withdecreased c-Myc protein levels in the mouse prostate tissueisolated from the LLC1 s.c. tumor model following either i.p. ori.t. AVI-4126 administration (Fig. 4). In contrast, AVI-4126bioavailability in the tumor core was minimal for the relativelyavascular PC-3 s.c. prostate xenograft tumors following i.p. AVI-4126 administration as shown in Fig. 5D. Immunoblot analysis(Fig. 5A) of the PC-3 tumor lysates revealed significantinhibition of c-Myc levels and AVI-4126 accumulation in thetumor following i.t. administration but not following i.p.administration. Table 1 summarizes the HPLC-based quantita-tive analysis of AVI-4126 PMO levels in the various experimen-tal tumor models. In situ tissue photomicrographs of the tumorcenter and periphery and flow cytometric analysis of theharvested tumor cells following i.p. or i.t. administration of afluorescent-tagged AVI-4126 PMO in the vascular LLC1 modelversus the avascular PC-3 tumor model showed a similar trend(Fig. 6).


Fig. 2. AVI-4126 bioavailability in LLC1s.c. syngeneic tumor. A, representativeimmunoblot from one of three studies (n = 6-8) showing probing of the tumorlysates with anti-c-Myc polyclonal antibody 24 hours post i.p. administration ofsaline or 300 Ag/mouseAVI-4126 PMO.The same blot was stripped and reprobedwith h-actin. Representative anion exchange HPLC chromatograms showingdetection of the oligomerAVI-4126 in control tumors (B) and AVI-4126 i.p. treatedtumor lysates (C).

Fig. 3. AVI-4126 bioavailability in LNCaP s.c. tumor xenograft. A, representativeimmunoblot from one study (n = 4) showing probing of the tumor lysates 24 hourspost i.p. administration of 300 Ag/mouse scrambled orAVI-4126 PMOwithanti-c-Myc polyclonal antibody.The same blot was stripped and reprobed withh-actin. Representative anion exchange HPLC chromatograms showing detectionof the oligomerAVI-4126 in control tumors (B) and AVI-4126 i.p. treated tumorlysates (C).




AVI-4126 plasma pharmacokinetics. Two separate studiesare being reported herein. One is a complete AVI-4126 plasmapharmacokinetic study in normal human subjects following asingle bolus i.v. dose of 90 mg (n = 5). Blood samples werecollected at various time points as indicated in Fig. 7. The secondstudy was conducted in patients undergoing resection surgeryfor prostate or breast tumors and the blood samples werecollected at 30 minutes and 24 hours following single i.v.administration of 90 mg AVI-4126 (n = 2). AVI-4126 PMOlevels in plasma levels were detectable up to the last time pointexamined following administration: 72 hours (the limit ofdetection of the analytic assay is 10 ng/mL). The data revealedabsence of metabolic breakdown as only full-length 20-mer testagent was recovered in plasma at all time points in both studies.

The data from both studies indicate a good correlation anda similar plasma concentration-time profile. The calculatedr2 value of the nonlinear regression analysis of the 90 mgdose group in the pharmacokinetic study (clinical study 1)was 0.8389 and changed to 0.8351 on inclusion of data fromthe bioavailability study (clinical study 2). The plasmaconcentration-time curve (Fig. 7) has at least two distinctphases. The curve has a relatively rapid distribution phasefollowed by a slower elimination phase. The pharmacokineticdata were best fit with a two-compartment model. Theanalysis revealed a volume of distribution equal to 266.1 F64.1 mL/kg and a clearance rate of 16.8 F 4.6 mL/h/kg. Thedistribution half-life for AVI-4126 was 1.0 F 0.1 hours,whereas the elimination half-life was 11.2 F 1.6 hours.

AVI-1426 bioavailability and safety in human solid tumors. A57-year-old woman with invasive ductal carcinoma of the

breast (T1NxM0, grade 3 of 3) and a 59-year-old man with anadenocarcinoma of the prostate (T3BNxM0, Gleason grade 3 +4) were given 90 mg (equivalent to 1 mg/kg according topatient weight) AVI-4126 by a slow i.v. push (f10-15


Fig. 4. AVI-4126 bioavailability in mouse prostate tissue isolated from the LLC1syngeneic s.c. tumor model as shown in Fig. 2. A, representative immunoblotshowingprobing of the tumor lysates with anti-c-Myc polyclonal antibody 24 hourspost-saline or100 Ag AVI-4126 PMO i.t. or 300 Ag i.p. administration.B, representative anion exchange HPLC chromatograms showing detection of theoligomerAVI-4126 in AVI-4126 i.p. treated prostate lysates.

Fig. 5. AVI-4126bioavailability inavascularPC-3 s.c. tumor xenograft.A, representative immunoblot fromoneof twostudies(n =6)showingprobingof thetumor lysateswithanti-c-Mycpolyclonalantibody24hourspost-salineor300AgAVI-4126 PMO i.t. or i.p. administration. Representative anion exchange HPLCchromatograms showing detection of the oligomerAVI-4126 in control tumors (B),AVI-4126 i.p. treated tumor lysates (C), and AVI-4126 i.t. treated tumor lysates (D).




seconds) 16 to 30 hours before the scheduled surgery. Noadverse events (graded according to the National CancerInstitute Common Toxicity Criteria) were observed during theadministration or at the follow-up periods. Tissue samplesthat included the core and margin of the tumor as well asadjacent normal tissue were collected from the resectionspecimens for measurements of the concentration anddistribution of AVI-4126. Representative photomicrographsof the breast and prostate tumors are shown in Fig. 8.Quantitation of AVI-4126 PMO levels in the tissue lysatesfrom the tumor margin, core, and adjacent normal tissue wascarried out by HPLC analysis. Table 2 summarizes the HPLC-based quantitative analysis of AVI-4126 PMO levels detectedin the resected prostate, breast tumor tissue, and plasma frompatients after a single i.v. administration of AVI-4126 PMO.

The data revealed significant tissue accumulation of AVI-4126in both breast and prostate tissues. The breast tumor hadincreased accumulation in the tumor core compared withmore uniform distribution in the surrounding normal tissue,tumor periphery, and tumor core in the prostate tumor.

Discussion

The current study provides a critical comparison of PMObioavailability between experimental animal models andhuman subjects in solid tumors following parenteral adminis-tration. We report here that PMO accumulation, dosing, andefficacy studies in animal tumor models can lead to reasonableprediction of human tumor bioavailability, although thenumber of patients studied is small.


Table1. Summary of HPLC-based quantitative analysis of AVI-4126 PMO levels detected in different tumor xenograftmodels or normalmouse prostate 24 hours after administration of AVI-4126 PMO

Model RouteDose(mg/kg)

PMOrecovered(Mg/g)

Tissueweightaverage (mg)

PMOrecovered(Mg/tissue)

PMOrecovered(% of total dose)

LLC1s.c. xenograft i.p. 15 0.62F 0.04 0.3 0.2 0.08DU145 s.c. xenograft i.p. 15 2.35 0.25 0.6 0.2LNCaP s.c. xenograft i.p. 15 2.85F 0.1 0.23 0.65 0.22PC-3 s.c. xenograft i.p. 15 Below detection limit 0.1 NA NA

i.t. 15 6F 3.7 0.08 0.48 0.16Mouse prostate i.p. 15 1.4 0.03 0.04 0.01

Fig. 6. Representative�200 tumorphotomicrographs of LLC1s.c. tumorxenograft 24 hours post i.p. (A) or i.t. (B)administration of fluorescein-labeledAVI-4126 (phase and fluorescent images ofidentical field). C, flow cytometric analysisto assess uptake of fluorescein-labeledAVI-4126 in LLC1tumor cells.Representative�200 tumorphotomicrographs of the tumor center (D)and tumor periphery (E) of PC-3 s.c. tumorxenograft 24 hours post i.p. administrationof fluorescein-labeled AVI-4126.Therespective phase and fluorescent imagesare from identical fields. F, flow cytometricanalysis to assess uptake offluorescein-labeled AVI-4126 in PC-3tumor cells.The representative xenografttumors in these studies were from earlierexperiments as explained in Figs. 2 and 5.




In vitro activity in a plasmid-based screening systemidentified an IC50 of 100 nmol/L for AVI-4126 c-MYCantisense PMO. The HPLC analysis revealed a concentrationof 720 ng/g in the breast tumor core, which represents a molarvalue of f103 nmol/L. This concentration is practicallyidentical to the IC50 value generated from the in vitro studies.These findings suggest that the dose tested produces tissueconcentrations of AVI-4126 in a human breast tumor thatwould be expected to inhibit the target. Not surprisingly, PMObioavailability increases with greater tumor vascularity inanimal models of cancer. The same relationship may holdtrue in human solid tumors (the breast carcinoma was

microscopically more vascular than the prostate cancer), butfurther testing is needed. The present study provided not onlynew information about AVI-4126 plasma pharmacokineticprofile in human subjects following i.v. administration butalso, by examining AVI-4126 in target tissue, suggests that thecurrent dose is pharmacodynamically relevant. Higher thanplasma concentration in the breast tumor suggests tissueaccumulation and may allow for less frequent dosing intervalthan dictated by plasma pharmacokinetics alone. For lessvascular tumors like prostate, frequent dosing or higher dosesmight be necessary.

c-MYC gene, the cellular homologue of the avian myelocyticleukemia virus, is implicated in a large number of human solidtumors, leukemias, and lymphomas as well as in a variety ofanimal neoplasias (25, 26). Overexpression of c-MYC associ-ated with uncontrolled cell proliferation is a frequent geneticevent in androgen-refractory adenocarcinoma of the prostate.Notably, androgen deprivation therapy has been observed toresult in c-MYC gene amplification associated with increasedcell proliferation (27, 28). Similarly, c-MYC is frequentlyoverexpressed in breast cancer and has been identified to beup-regulated by estrogen stimulation of hormone-dependentbreast cancer cells (29). Thus, an inhibition strategy for c-MYCis likely to provide an attractive therapeutic modality formultiple neoplastic settings (2).

The application of antisense strategy (30) as therapeuticagents in oncology has come a long way in the last two decadeswith various agents currently in clinical trials (31–33). Themost widely used antisense chemistry includes the phosphor-othioates, where the oligonucleotide backbone is stabilized bythe substitution of sulfur for oxygen at the phosphorous of the


Fig. 7. Plasma concentration-time curve of AVI-4126 following single bolus i.v.administration of 90 mg in human test subjects.The data points in study1are fromblood samples collected at various time points from normal human subjectsfollowing single i.v. AVI-4126 administration (n =5 per timepoint).Those from study2 are blood samples collected at 30 minutes and 24 hours post i.v. administrationof AVI-4126 in patients undergoing prostate or breast resection surgery (n = 2).

Fig. 8. Representative photomicrographsof the resected breast and prostate tumors.At low power (original magnification,�40,left), the breast carcinoma shows areas ofcentral necrosis and fibrosis. A closer viewof the tumor edge (�100, right) revealsnumerous capillaries (arrowheads) and amedium-sized vein (arrow).A medium-power image of the prostateadenocarcinoma (�100, left) showsperineural invasion and extension of thetumor into adjacent fat.The architecture ofthe glands viewed at higher power(�200, right) consists of a mixture ofintermediate-grade (Gleason pattern 3)and higher-grade (Gleason pattern 4)patterns.




molecule (34). The first and only antisense drug (Formivirsen,ISIS Pharmaceuticals, Inc., Carlsbad, CA) approved by the U.S.Food and Drug Administration is a phosphorothioate used forthe treatment of cytomegalovirus-induced retinitis in AIDSpatients. Although phosphorothioates are relatively resistant toenzymatic degradation and exhibit greater nuclease stabilitythan the phosphodiester oligonucleotides, they are digestedintracellularly (35). The phosphorothioates have also beenshown to nonspecifically bind to cellular components andthese interactions potentially interfere with the translationalprocess (36, 37). On the other hand, PMO chemistry andunique RNase H–independent mechanism of action seem tooffer stable, nontoxic, and specific translational inhibitors.Functional efficacy of multiple PMO antisense agents targetingvarious genes, including c-MYC, have been shown in preclinicalcancer models (10, 38–40). I.t. administration of AVI-4126 inthe avascular PC-3 xenograft tumors decreased tumor burdenby f75% compared with control treatments (3). In a Lewislung carcinoma murine model, a combination regimen inwhich cisplatin was given on days 2 to 4 and 13 to 15 followedby AVI-4126 treatment on days 6 to 12 and 17 to 23 inhibitedtumor growth significantly in comparison with cisplatin alone(17). Similar potentiation of antitumor activity of AVI-4126 byi.p. administration was also observed in a novel combinationstrategy involving inhibition of both c-MYC and h-humanchorionic gonadotropin using PMOs in a prostate cancer model

(18). Thus, AVI-4126 is a promising new agent that might beused alone or in combination with either hormonal orcytotoxic therapies in solid tumors.

AVI-4126 has been tested in multiple clinical trials andshown to have an excellent safety profile. The commonlyobserved side effects like increase in APTT, complement C3a,anemia, decrease in platelet count, and hypotension related tovarious phosphorothioates (35, 41, 42) were not observedwith the AVI-4126 PMO in human clinical trials via bolus i.v.,local, and s.c. routes of administration. Pharmacokineticstudies in human subjects and animal models have alsoestablished the feasibility of once-daily dosing (16). Currently,efficacy of AVI-4126 is being tested as a cancer therapeutic inclinical studies, which will include not only assessment oftissue PMO concentration but also, whenever feasible,assessment of target expression before and after administra-tion of AVI-4126. In summary, this is the first study that hascharacterized PMO bioavailability in solid tumors and revealsthe potential of PMOs as a clinically viable strategy in cancertherapeutics.

Acknowledgments

We thank Dr. Gene F. Ray (AAI Development Services, Shawnee, KS) for theHPLC analysis of PMO in human tissue, the biology, synthesis, analytic, and clinicalgroups at AVI, and Dianne Lemmon, RN, for expert patient care during this study.


Table 2. Summary of HPLC-based quantitative analysis of AVI-4126 PMO levels detected in the resected prostate,breast tumor tissue, and plasma from patients 24 hours after i.v. administration of AVI-4126 PMO

Tumor RouteAVI-4126dose (mg)

Patientweight (lb)

PMOrecoveredin surroundingnormal tissue(ng/g)

PMOrecoveredin tumor periphery(ng/g)

PMOrecoveredin tumor core(ng/g)

PMOrecoveredin plasma(24 h; ng/mL)

Breast stage I T1NxG3M0 i.v. 90 182 110 255.4 720 700Prostate stage I T2NxM0 i.v. 90 209 286.2 168.4 179.9 395

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2005;11:3930-3938. Clin Cancer Res Gayathri R. Devi, Tomasz M. Beer, Christopher L. Corless, et al. AVI-4126, in Solid TumorsAntisense Phosphorodiamidate Morpholino Oligomer,

c-MYC Bioavailability and Pharmacokinetics of a In vivo

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