Cancer Therapy: Preclinical

In vivo Bioavailability and Pharmacokinetics of a c-MYC Antisense Phosphorodiamidate Oligomer, AVI-4126, in Solid Tumors Gayathri R. Devi,1 Tomasz M. Beer,2 Christopher L. Corless,2 Vikram Arora,1 Doreen L.Weller,1andPatrick L. Iversen 1

Abstract Phosphorodiamidate morpholino oligomers (PMO) inhibit targeted gene expression by prevent- ing ribosomal assembly, thereby preventing mRNA translation. AVI-4126, a PMO targetedagainst c-MYC, has been extensively characterizedin multiple andother disease models andis currently in human clinical trials. A phase I clinical study was conducted to address the issue of PMO bioavailability in malignant tumors surgically excisedfrom patients with adenocarcinoma of prostate andbreast 1day after i.v. administration of a single dose of 90 mg AVI-4126 PMO. The study objectives were to evaluate safety, to determine AVI-4126 concentration in tissue samples of the tumors, andto examine the distribution of AVI-4126(margin versus tumor core).Significant concentrations of intact PMO similar to the animal models were detected in both human prostate andbreast tumor tissues with increaseddistribution in the tumor core for the vascular breast tumors. No serious adverse events (graded according to National Cancer Institute Common Toxi- city Criteria) were reported. Another phase I study was conducted in normal human volunteers to assess AVI-4126plasma pharmacokinetics following single i.v. administration of 90 mg AVI-4126. Data from both human studies indicated similar plasma concentration-time profile. These studies show PMO bioavailability in tumor tissue andestablish the feasibility of using PMO targeting spe- cific genes in human cancer clinical trials.

The c-MYC proto-oncogene, which is frequently overex- istry avoids a variety of potential significant limitations, pressed in human cancer, plays a critical role in the control including the binding of other cellular and extracellular of cell proliferation, differentiation, and apoptosis (1, 2). proteins that is observed with charged chem- Several different strategies, including antisense-mediated istries (8, 9). inhibition of the gene, have been employed to develop The 20-mer AVI-4126 sequence was chosen based on novel drugs that seek to inactivate c-MYC for the treatment favorable solubility, efficacy, and potency compared with of cancer (2). AVI-4126, a phosphorodiamidate morpholino >100 other antisense c-MYC PMOs targeted to various sites oligomer (PMO) targeting c-MYC, has an excellent safety along the c-MYC 5V untranslated region, the splice acceptor of profile in preclinical animal models and in human clinical the first intron, and around the translation start site. Previous trials (3). PMOs are neutrally charged antisense agents, studies have shown that AVI-4126 inhibits c-MYC translation wherein the deoxyribose moiety of DNA is replaced with in a sequence-specific manner in cancer cell lines in culture by a six-membered morpholine ring and the charged phospho- steric blockade and missplicing of pre-mRNA, resulting in diester internucleoside linkage is replaced with phosphoro- significant growth inhibition (10, 11). AVI-4126 has been diamidate linkages (4, 5). PMO antisense molecules are steric observed to specifically down-regulate c-Myc protein, decrease blockers and inhibit gene expression by physically preventing cyst formation, and improve renal function in a murine model binding or progression of splicing or translational machinery of polycyctic kidney disease (12); causes growth arrest in a components (5–7). The neutral character of the PMO chem- rat liver regeneration model (13); and is effective in prevent- ing cardiac restenosis in multiple animal models (14, 15). The safety profile of the PMO chemistry, including that of AVI- 4126, has been widely studied in animal toxicity models as Authors’ Affiliations: 1AVIBioPharma, Inc., Corvallis, Oregon and 2Oregon Health well as in human clinical trials after systemic and local routes andScience University Cancer Institute, Portland,Oregon of administration (3, 16). No serious side effects (graded ac- Received10/13/04; revised2/16/05; accepted2/18/05. cording to the National Cancer Institute Common Toxicity Grant support: AVI BioPharma, Inc. andDepartment of Defense grant DAMD17- 01-0017 (G.R. Devi). Criteria) have been reported. AVI-4126 antisense PMO has also The costs of publication of this article were defrayed in part by the payment of page shown promising efficacy in tumor models of lung cancer (17) charges. This article must therefore be hereby marked advertisement in accordance and (3, 18, 19). Therefore, the purpose of this with 18 U.S.C. Section 1734 solely to indicate this fact. study was to characterize PMO bioavailability in solid tumor Requests for reprints: Gayathri R. Devi, Comprehensive Cancer Center, Duke models in animals and in surgically excised malignant tumors University Medical Center, 401 MSRB, Box 2606, Durham, NC 27710. Phone: 919-668-0410; Fax: 919-681-7970; E-mail: [email protected]. from patients after single systemic AVI-4126 administration F 2005 American Association for Cancer Research. and to assess pharmacokinetics and safety.

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Materials and Methods i.p. as indicated. After 24 hours, the animals were euthanized and tumors were excised and processed for flow cytometry, photomicrog- Oligomers raphy, histology, and/or preparation of lysates. AVI-4126 (c-MYC antisense PMO; 5V-ACGTTGAGGGGCATCGTCGC- Protein expression in mouse tumors 3V) and control scrambled (5V-ACTGTGAGGGCGATCGCTGC-3V) mor- Tumor samples were lysed immediately after the animal was pholino oligomers were synthesized at AVI BioPharma, Inc. (Corvallis, euthanized. Protein estimation was carried out by Bradford protein OR) as described previously (4, 18). Purity was >95% as determined by assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein in cell or reverse-phase high-performance liquid chromatography (HPLC) and tissue lysates were subjected to 10% or 12% SDS-PAGE under reducing matrix-assisted laser desorption ionization time of flight mass conditions and probed with anti c-Myc polyclonal antibody as spectroscopy. described previously (18).

Plasmid-based test system for screening Photomicrography of mouse tumors phosphorodiamidate morpholino oligomer antisense Tumor slices were placed in plastic molds filled with embedding activity medium for frozen tissue processing (Sakura Finetek, Torrance, CA). Afusionc-MYC luciferase construct (pCiNeo-myc-LucDA) was The molds were wrapped in aluminum foil to protect from light and generated in the pCiNeo expression vector (Promega, Madison, WI) frozen in a 80jC freezer. Cryostat sections (5 Am) were cut at Oregon as described previously (20). This plasmid features a T7 promoter capable State University Veterinary Diagnostic Laboratory. Slides were air dried of generating in vitro transcribed RNA from a cloned insert for use in cell- in the dark for 30 minutes and coverslip mounted using Fluoromount- free rabbit reticulocyte in vitro translation reactions and a cytomegalo- G mounting medium (Southern Biotechnology Associates, Birming- virus promoter for constitutive expression in mammalian cells. In vitro ham, AL). The photomicrographs were taken with a Nikon Diaphot 300 transcription was carried out with T7 Mega script (Ambion, Austin, TX). microscope connected to an Olympus (Melville, NY) Magnafire SP- brand digital camera. The exposure times were kept constant for all Cell-free luciferase assay fluorescent pictures at 25 seconds. In vitro translation was done by mixing rabbit reticulocyte lysate with known amounts of antisense, scrambled PMOs or vehicle (water) Flow cytometry of mouse tumor cells f followed by addition of a known amount of the c-MYC/Luc RNA ( 1 Tumor cells from animals injected with fluorescein-labeled AVI- nmol/L final concentration). The Promega luciferase assay reagent 4126 were harvested by digestion with 0.1% collagenase (type I) and protocol was followed. The percent inhibition of luciferase activity 50 mg/mL DNase as described previously (21). Dispersed cells were compared with control was calculated based on readings from a washed thrice with PBS and resuspended in fluorescence-activated cell luminometer (Cardinal, Santa Fe, NM). sorting buffer. Flow cytometry was carried out at the Oregon State University Core Facility using the BD FACSCalibur cytometer (Becton Cell culture Dickinson, Mountain View, CA) and data were analyzed using the FCS PC-3, LNCaP, DU145, human prostate cancer cell lines, and LLC1 express software. murine Lewis lung cancer cell line were obtained from American Type Culture Collection (Rockville, MD). The prostate cancer cells were High-performance liquid chromatography detection of cultured in RPMI 1640 (Hyclone Laboratories, Logan, UT) supple- phosphorodiamidate morpholino oligomer in tumor mented with 10% fetal bovine serum (Life Technologies, Gaithersburg, and organ tissue MD), 100 units/mL penicillin, and 75 units/mL streptomycin at 37jC Tumor tissue, liver, and kidney lysates from AVI-4126-treated under 5% CO . LLC1 were maintained in DMEF-12 medium (Hyclone 2 animals were prepared as described above and analyzed for presence Laboratories) supplemented with 10% fetal bovine serum, 100 units/mL of PMO by HPLC analysis as described (22). Briefly, a 10-AL aliquot penicillin, 100 Ag/mL streptomycin, and 0.25 Ag/mL amphotericin. (500 ng) of the internal standard PMO (15-mer whose sequence was Animals derived from a 5V truncation of AVI-4126) was added to all 250-AL Male athymic (Ncr nu/nu) nude mice and C57BL/6 mice f4 weeks aliquots of tumor, kidney, and liver lysate (0.2 g/mL) samples. A old on arrival were obtained from Simonson Laboratories (Gilroy, CA) Methanol (300 L) was added to each sample and the tubes were and were housed in sterile plastic cages at the Laboratory Animal vortexed. The tubes were centrifuged for 10 minutes using a high-speed Resources Facility at Oregon State University (Corvallis, OR). The centrifuge and supernatants were transferred to new Eppendorf tubes. athymic mice were housed in laminar airflow cabinets in microisolator The pellet was then washed with 100 AL Tris and the wash buffer was cages under pathogen-free conditions with a 12-hour light/dark schedule added to the supernatant. The supernatants were heated in a water bath j and fed autoclaved standard chow and water ad libitum. LNCaP and PC-3 at 70 C for 10 minutes. The samples were recentrifuged for 10 minutes cells (2 106) were suspended in 150 AL RPMI 1640 plus 10% fetal and the supernatants were transferred to new Eppendorf tubes. bovine serum and 150 AL Matrigel (Becton Dickinson, Palo Alto, CA). Methanol was evaporated using a Speed vac (Savant, Farmingdale, DU145 cells (5 106) were suspended in 150 AL RPMI 1640 and injected NY) and the samples were finally transferred to clear shell vials and via a 25-gauge needle into the s.c. space of the flank region of 5- to 6- lyophilized after the addition of 100 AL deionized water to each vial. week-old athymic male mice (3, 18). LLC1 cells (2 105) were injected The lyophilized samples were reconstituted using 100-AL aliquots of 5V s.c. into the right rear flank of the C57BL/6 mice (17). All animals were fluoresceinated DNA (1.0 absorbance units/mL) whose sequence was anesthetized with isofluorane before injection of cells. All animal complementary to that of AVI-4126 PMO. A set of AVI-4126 standards protocols conformed to the ethical guidelines of the 1975 Declaration was prepared by spiking the PMO into 250-AL aliquots of blank rat of Helsinki and were approved by the Institutional Animal Care and Use plasma (10, 25, 50, 100, 250, 500, and 1,000 ng/250 AL plasma) along Committee of Oregon State University. with the internal standard. The standards were extracted similarly. The samples were analyzed by injecting onto a Dionex DNA Pac Mouse treatment protocols PA-100 column (4 250 mm column, Dionex Corp., Sunnyvale, CA) The experiments were started when the tumors of each type of using a Varian autosampler (AI-200) connected to a Varian HPLC xenografts were well established and the tumor volumes were between pump (model 9010 inert) equipped with a Varian fluorescence 500 and 700 mm3, and the mice were randomly assigned to different detector (model 9075). The mobile phases [A: 0.025 mol/L Tris-HCl groups (n = 4-8). AVI-4126 PMO (300 Ag equivalent to a dose of (pH 8); B: 0.025 mol/L Tris (pH 8)/1.0 mol/L NaCl] were prepared 15 mg/kg) dissolved in saline was given either intratumorally (i.t.) or using HPLC-grade water and reagents and filtered through a 0.2 Am

www.aacrjournals.org 3931 Clin Cancer Res 2005;11(10) May 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical filter before use. The gradient program employed was [90-10% B] at before AVI-4126 dosing, 30 minutes later, before anesthesia, and after 0 minute and [55-45% B] at 20 minutes while the pump was held at a anesthesia recovery. Adverse events were recorded 30 minutes after flow rate of 1.5 mL/min. The runs were monitored at excitation and dosing and again 7 to 14 days later. Tissue specimens that included the emission wavelengths of 494 and 518 nm, respectively. core and margin of the tumor as well as adjacent normal tissue were collected with the goal of measurements of the concentration and Pharmacokinetic study distribution of AVI-4126. A 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 Results specifications and a detailed time and events schedule were prepared by MDS Harris (Lincoln, NE) with local ethics committee approval. Five AVI-4126 activity in a plasmid-based in vitro screening system. dose levels were evaluated with six normal healthy subjects enrolled in To ascertain the sequence specificity of the PMO, AVI-4126, a each cohort after screening medical history, examination, and plasmid-based test system was used for cell-free screening. A laboratory tests had shown no clinically significant abnormalities. fusion construct, pCiNeo-myc-LucDA, was generated by sub- Cohorts 1 (three males and three females), 2 (two males, four females), cloning a small segment of the c-MYC gene that includes the 3 (three males, three females), 4 (two males, four females), and 5 (four AUG translation start site followed by luciferase into the males and two females) received 1, 3, 10, 30, and 90 mg AVI-4126, pCiNeo expression vector. In vitro translation was done by respectively, given as a slow (10-15 seconds) i.v. bolus/push. The mixing the rabbit reticulocyte lysate with known amounts of subjects in all five dose levels were in the fed state. They were confined antisense, scrambled oligomers or vehicle (water) followed by to the clinic through the 72-hour post-dose events. The pharmacoki- addition of a known amount of the c-MYC/Luc in vitro netic variables were studied for the highest dose (90 mg). Blood draw transcribed RNA. The relative inhibition of luciferase activity was conducted at the following time points: 0.033, 0.083, 0.167, 0.25, in the presence of various concentrations of PMO compared 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hours following AVI-4126 administration. with the vehicle control was calculated based on the readings from a luminometer. The data in Fig. 1 reveal a dose- Pharmacokinetic analysis dependent and specific inhibition of luciferase activity, IC50 of The data were evaluated with the assistance of the computer program 100 nmol/L in the presence of increasing concentrations of PKCALC as published previously (23). The i.v. plasma concentrations the antisense c-MYC PMO (AVI-4126) compared with the measured were fit to Eq. A (24): scrambled PMO control. Differential bioavailability of AVI-4126 in murine solid tumors t t CðtÞ¼Ae þ Be ðAÞ models. Three different tumor models with varying degrees of where C(t) is the plasma concentration at time t, A and B are vascularity (LLC1 syngeneic lung tumor, LNCaP prostate intercept terms, a is a distribution rate constant, and h is an xenograft, and PC-3 prostate xenograft) were employed to elimination rate constant. All data are expressed as mean F SE as explore the relationship between tumor blood supply and determined by the computer program InStat 2 (GraphPad, San Diego, accumulation of AVI-4126 in the tumor. In each case, a single CA). Graphs were prepared with Prism version 3.0 (GraphPad). dose (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, Phase I study of AVI-4126 localization in human solid tumors In 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 or histologically confirmed adenocarcinoma of the breast, colon, prostate, or non–small cell lung cancer who had a tumor at least 1.0 1.0 cm and were scheduled for surgical resection were eligible. Patients could not have metastases, active infection, or another serious medical condition; could not be pregnant or nursing; could not have received an investigational agent within 30 days or any prior chemotherapy, hormonal therapy, or radiation therapy; and had to have adequate hematologic, renal, and hepatic function (hemoglobin >11 g/dL for men and >10 g/dL for women; serum creatinine V institutional upper limits of normal; serum bilirubin < upper limits of normal; aspartate aminotransferase < 1.5 upper limits of normal). All patients provided signed informed consent and the study was approved by the Institutional Review Board of the Oregon Health and 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) f Fig. 1. Plasmid-based test system for screening AVI-4126 PMO sequence was given by a slow i.v. push ( 10-15 seconds) 16 to 30 hours before specificity andantisense inhibition of c-MYC mRNA in the region from 5V the scheduled surgery. Vital signs were monitored before and untranslatedregion in to the AUG translation start site with the luciferase reporter 30 minutes after dosing, before hospital discharge, and 7 to 14 days gene. In vitro translation was done by mixing rabbit reticulocyte lysate with various later. Complete blood count and serum chemistries were obtained concentrations of vehicle (water), antisense, or scrambledPMOs in the presence of c-MYC luciferase RNA. Points, mean (n = 3) to reveal the luciferase light units in the before hospital discharge and again 7 to 14 days later. EDTA-stabilized lysates normalizedfor protein content to show specificity of the antisense c-MYC plasma was collected for AVI-4126 concentration analysis immediately PMO in comparison with a 70% identical but scrambled sequence; bars, SE.

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neither was detected in the tissue or tumor lysates. Significant levels of AVI-4126 PMO were detected, which correlated with decreased c-Myc protein levels in the mouse prostate tissue isolated from the LLC1 s.c. tumor model following either i.p. or i.t. AVI-4126 administration (Fig. 4). In contrast, AVI-4126 bioavailability in the tumor core was minimal for the relatively avascular 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 significant inhibition of c-Myc levels and AVI-4126 accumulation in the tumor 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 tumor center and periphery and flow cytometric analysis of the harvested tumor cells following i.p. or i.t. administration of a fluorescent-tagged AVI-4126 PMO in the vascular LLC1 model versus the avascular PC-3 tumor model showed a similar trend (Fig. 6).

Fig. 2. AVI-4126 bioavailability in LLC1s.c. syngeneic tumor. A, representative immunoblot from one of three studies (n = 6-8) showing probing of the tumor lysates with anti-c-Myc polyclonal antibody 24 hours post i.p. administration of saline or 300 Ag/mouse AVI-4126 PMO. The same blot was strippedandreprobed with h-actin. Representative anion exchange HPLC chromatograms showing detection of the oligomer AVI-4126 in control tumors (B) andAVI-4126 i.p. treated tumor lysates (C). scrambled PMO, or saline administration and subjected to flow cytometric analysis, preparation of cryosections, and tumor lysates. Significant AVI-4126 bioavailability was detected up to 24 hours after a single i.p. administration in the LLC1 lung syngeneic tumors (Fig. 2) and LNCaP prostate xenograft tumors, both of which are highly vascular (Fig. 3) Immunoblot analysis of the respective tumor lysates (Figs. 2A and 3A) revealed specific inhibition of target c-Myc protein levels in the AVI-4126 PMO-treated tumors compared with the control groups. Quantitation of AVI-4126 PMO levels in the tissue lysates from the tumors was carried out by HPLC analysis. The elution order of each chromatogram is AVI-4126, the internal standard, and the excess fluoresceinated DNA probe. The peak Fig. 3. AVI-4126 bioavailability in LNCaP s.c. tumor xenograft. A, representative corresponding to full-length AVI-4126 was readily observed in immunoblot from one study (n = 4) showing probing of the tumor lysates 24 hours both LLC1 (Fig. 2B and C) and LNCaP (Fig. 3B and C) tumor post i.p. administration of 300 Ag/mouse scrambledorAVI-4126 PMO with tissue samples as shown in the representative chromatograms. anti-c-Myc polyclonal antibody.The same blot was stripped and reprobed with h-actin. Representative anion exchange HPLC chromatograms showing detection This analytic technique is capable of resolving peaks resulting of the oligomerAVI-4126 in control tumors (B) andAVI-4126 i.p. treatedtumor from (N - 1)-mers and truncated versions of AVI-4126, but lysates (C).

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

Fig. 4. AVI-4126 bioavailability in mouse prostate tissue isolatedfrom the LLC1 syngeneic s.c. tumor model as shown in Fig. 2. A, representative immunoblot showing probing of the tumor lysates with anti-c-Myc polyclonal antibody 24 hours post-saline or100 Ag AVI-4126 PMO i.t. or 300 Ag i.p. administration. B, representative anion exchange HPLC chromatograms showing detection of the oligomerAVI-4126 in AVI-4126 i.p. treatedprostate lysates.

AVI-4126 plasma pharmacokinetics. Two separate studies are being reported herein. One is a complete AVI-4126 plasma pharmacokinetic study in normal human subjects following a single bolus i.v. dose of 90 mg (n = 5). Blood samples were collected at various time points as indicated in Fig. 7. The second study was conducted in patients undergoing resection surgery for prostate or breast tumors and the blood samples were collected at 30 minutes and 24 hours following single i.v. administration of 90 mg AVI-4126 (n = 2). AVI-4126 PMO levels in plasma levels were detectable up to the last time point examined following administration: 72 hours (the limit of detection of the analytic assay is 10 ng/mL). The data revealed absence of metabolic breakdown as only full-length 20-mer test agent was recovered in plasma at all time points in both studies. The data from both studies indicate a good correlation and a similar plasma concentration-time profile. The calculated r2 value of the nonlinear regression analysis of the 90 mg dose group in the pharmacokinetic study (clinical study 1) was 0.8389 and changed to 0.8351 on inclusion of data from the bioavailability study (clinical study 2). The plasma concentration-time curve (Fig. 7) has at least two distinct phases. The curve has a relatively rapid distribution phase followed by a slower elimination phase. The pharmacokinetic data were best fit with a two-compartment model. The analysis revealed a volume of distribution equal to 266.1 F F 64.1 mL/kg and a clearance rate of 16.8 4.6 mL/h/kg. The Fig. 5. AVI-4126bioavailability in avascular PC-3 s.c. tumor xenograft. distribution half-life for AVI-4126 was 1.0 F 0.1 hours, A, representativeimmunoblot fromone of two studies (n = 6) showing probing of the whereas the elimination half-life was 11.2 F 1.6 hours. tumorlysates with anti-c-Myc polyclonalantibody 24 hours post-saline or 300 Ag AVI-4126 PMO i.t. or i.p. administration. Representative anion exchange HPLC AVI-1426 bioavailability and safety in human solid tumors. A chromatograms showing detection of the oligomer AVI-4126 in control tumors (B), 57-year-old woman with invasive ductal carcinoma of the AVI-4126 i.p. treatedtumor lysates ( C), andAVI-4126 i.t. treatedtumor lysates ( D).

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Ta b l e 1. Summary of HPLC-basedquantitative analysis of AVI-4126PMO levels detectedin different tumor xenograft models or normal mouse prostate 24 hours after administration of AVI-4126 PMO

Dose PMO recovered Tissue weight PMO recovered PMO recovered Model Route (mg/kg) (Mg/g) average (mg) (Mg/tissue) (% of total dose)

LLC1s.c. xenograft i.p. 15 0.62 F 0.04 0.3 0.2 0.08 DU145 s.c. xenograft i.p. 15 2.35 0.25 0.6 0.2 LNCaP s.c. xenograft i.p. 15 2.85 F 0.1 0.23 0.65 0.22 PC-3 s.c. xenograft i.p. 15 Below detection limit 0.1 NA NA i.t. 15 6 F 3.7 0.08 0.48 0.16 Mouse prostate i.p. 15 1.4 0.03 0.04 0.01

seconds) 16 to 30 hours before the scheduled surgery. No The data revealed significant tissue accumulation of AVI-4126 adverse events (graded according to the National Cancer in both breast and prostate tissues. The breast tumor had Institute Common Toxicity Criteria) were observed during the increased accumulation in the tumor core compared with administration or at the follow-up periods. Tissue samples more uniform distribution in the surrounding normal tissue, that included the core and margin of the tumor as well as tumor periphery, and tumor core in the prostate tumor. adjacent normal tissue were collected from the resection specimens for measurements of the concentration and Discussion distribution of AVI-4126. Representative photomicrographs of the breast and prostate tumors are shown in Fig. 8. The current study provides a critical comparison of PMO Quantitation of AVI-4126 PMO levels in the tissue lysates bioavailability between experimental animal models and from the tumor margin, core, and adjacent normal tissue was human subjects in solid tumors following parenteral adminis- carried out by HPLC analysis. Table 2 summarizes the HPLC- tration. We report here that PMO accumulation, dosing, and based quantitative analysis of AVI-4126 PMO levels detected efficacy studies in animal tumor models can lead to reasonable in the resected prostate, breast tumor tissue, and plasma from prediction of human tumor bioavailability, although the patients after a single i.v. administration of AVI-4126 PMO. number of patients studied is small.

Fig. 6. Representative 200 tumor photomicrographs of LLC1s.c. tumor xenograft 24 hours post i.p. (A)ori.t.(B) administration of fluorescein-labeled AVI-4126 (phase andfluorescent images of identical field). C, flow cytometric analysis to assess uptake of fluorescein-labeled AVI-4126 in LLC1tumor cells. Representative 200 tumor photomicrographs of the tumor center (D) andtumor periphery ( E)ofPC-3s.c.tumor xenograft 24 hours post i.p. administration of fluorescein-labeledAVI-4126.The respective phase andfluorescent images are from identical fields. F, flow cytometric analysis to assess uptake of fluorescein-labeledAVI-4126 in PC-3 tumor cells.The representative xenograft tumors in these studies were from earlier experiments as explainedin Figs. 2 and5.

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microscopically more vascular than the prostate cancer), but further testing is needed. The present study provided not only new information about AVI-4126 plasma pharmacokinetic profile in human subjects following i.v. administration but also, by examining AVI-4126 in target tissue, suggests that the current dose is pharmacodynamically relevant. Higher than plasma concentration in the breast tumor suggests tissue accumulation and may allow for less frequent dosing interval than dictated by plasma pharmacokinetics alone. For less vascular tumors like prostate, frequent dosing or higher doses might be necessary. c-MYC gene, the cellular homologue of the avian myelocytic leukemia virus, is implicated in a large number of human solid 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 from tumors, leukemias, and lymphomas as well as in a variety of bloodsamples collectedat various time points from normal human subjects animal neoplasias (25, 26). Overexpression of c-MYC associ- following single i.v. AVI-4126administration (n = 5 per time point).Those from study ated with uncontrolled cell proliferation is a frequent genetic 2 are bloodsamples collectedat 30 minutes and24 hours post i.v. administration of AVI-4126 in patients undergoing prostate or breast resection surgery (n =2). event in androgen-refractory adenocarcinoma of the prostate. Notably, androgen deprivation therapy has been observed to result in c-MYC gene amplification associated with increased In vitro activity in a plasmid-based screening system cell proliferation (27, 28). Similarly, c-MYC is frequently identified an IC50 of 100 nmol/L for AVI-4126 c-MYC overexpressed in breast cancer and has been identified to be antisense PMO. The HPLC analysis revealed a concentration up-regulated by estrogen stimulation of hormone-dependent of 720 ng/g in the breast tumor core, which represents a molar breast cancer cells (29). Thus, an inhibition strategy for c-MYC value of f103 nmol/L. This concentration is practically is likely to provide an attractive therapeutic modality for identical to the IC50 value generated from the in vitro studies. multiple neoplastic settings (2). These findings suggest that the dose tested produces tissue The application of antisense strategy (30) as therapeutic concentrations of AVI-4126 in a human breast tumor that agents in oncology has come a long way in the last two decades would be expected to inhibit the target. Not surprisingly, PMO with various agents currently in clinical trials (31–33). The bioavailability increases with greater tumor vascularity in most widely used antisense chemistry includes the phosphor- animal models of cancer. The same relationship may hold othioates, where the oligonucleotide backbone is stabilized by true in human solid tumors (the breast carcinoma was the substitution of sulfur for oxygen at the phosphorous of the

Fig. 8. Representative photomicrographs of the resectedbreast andprostate tumors. At low power (original magnification, 40, left), the breast carcinoma shows areas of central necrosis andfibrosis. A closer view of the tumor edge (10 0, right)reveals numerous capillaries (arrowheads)anda medium-sized vein (arrow). A medium-power image of the prostate adenocarcinoma (100, left)shows perineural invasion andextension of the tumor into adjacent fat.The architecture of the glands viewed at higher power (200, right) consists of a mixture of intermediate-grade (Gleason pattern 3) andhigher-grade(Gleason pattern 4) patterns.

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Ta b l e 2. Summary of HPLC-basedquantitative analysis of AVI-4126 PMO levels detectedin the resectedprostate, breast tumor tissue, andplasma from patients 24 hours after i.v. administration of AVI-4126 PMO

PMO recovered in surrounding PMO recovered PMO recovered PMO recovered AVI-4126 Patient normal tissue in tumor periphery in tumor core in plasma Tumor Route dose (mg) weight (lb) (ng/g) (ng/g) (ng/g) (24 h; ng/mL)

Breast stage I T1NxG3M0 i.v. 90 182 110 255.4 720 700

Prostate stage I T2NxM0 i.v. 90 209 286.2 168.4 179.9 395

molecule (34). The first and only antisense drug (Formivirsen, (18). Thus, AVI-4126 is a promising new agent that might be ISIS Pharmaceuticals, Inc., Carlsbad, CA) approved by the U.S. used alone or in combination with either hormonal or Food and Drug Administration is a phosphorothioate used for cytotoxic therapies in solid tumors. the treatment of cytomegalovirus-induced retinitis in AIDS AVI-4126 has been tested in multiple clinical trials and patients. Although phosphorothioates are relatively resistant to shown to have an excellent safety profile. The commonly enzymatic degradation and exhibit greater nuclease stability observed side effects like increase in APTT, complement C3a, than the phosphodiester , they are digested anemia, decrease in platelet count, and hypotension related to intracellularly (35). The phosphorothioates have also been various phosphorothioates (35, 41, 42) were not observed shown to nonspecifically bind to cellular components and with the AVI-4126 PMO in human clinical trials via bolus i.v., these interactions potentially interfere with the translational local, and s.c. routes of administration. Pharmacokinetic process (36, 37). On the other hand, PMO chemistry and studies in human subjects and animal models have also unique RNase H–independent mechanism of action seem to established the feasibility of once-daily dosing (16). Currently, offer stable, nontoxic, and specific translational inhibitors. efficacy of AVI-4126 is being tested as a cancer therapeutic in Functional efficacy of multiple PMO antisense agents targeting clinical studies, which will include not only assessment of various genes, including c-MYC, have been shown in preclinical tissue PMO concentration but also, whenever feasible, cancer models (10, 38–40). I.t. administration of AVI-4126 in assessment of target expression before and after administra- the avascular PC-3 xenograft tumors decreased tumor burden tion of AVI-4126. In summary, this is the first study that has by f75% compared with control treatments (3). In a Lewis characterized PMO bioavailability in solid tumors and reveals lung carcinoma murine model, a combination regimen in the potential of PMOs as a clinically viable strategy in cancer which cisplatin was given on days 2 to 4 and 13 to 15 followed therapeutics. by AVI-4126 treatment on days 6 to 12 and 17 to 23 inhibited tumor growth significantly in comparison with cisplatin alone (17). Similar potentiation of antitumor activity of AVI-4126 by Acknowledgments i.p. administration was also observed in a novel combination We thank Dr. Gene F. Ray (AAI Development Services, Shawnee, KS) for the strategy involving inhibition of both c-MYC and h-human HPLC analysis of PMO in human tissue, the biology, synthesis, analytic, andclinical chorionic gonadotropin using PMOs in a prostate cancer model groups at AVI, andDianne Lemmon, RN, for expert patient care during this study.

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Gayathri R. Devi, Tomasz M. Beer, Christopher L. Corless, et al.

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