Tumor-specific antibody-mediated targeted delivery of Doxil® reduces the manifestation of auricular erythema side effect in mice

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Tumor-specific antibody-mediated targeted delivery of Doxil® reduces the manifestation of auricular erythema side effect in mice
  Tumor-Specific Antibody-Mediated Targeted Delivery of Doxil ® Reduces the Manifestation of Auricular Erythema Side Effect inMice Tamer A. Elbayoumi † and Vladimir P. Torchilin * Department of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology andNanomedicine, Northeastern University, Boston, MA 02115, USA  Abstract A mucocutaneous reaction in mice associated with Doxil ®  treatment was identified as Auricular Erythema (AE). Given that the immuno-targeting of Doxil ®  to tumors was found to influence alsoits systemic biodistribution pattern, the attempt was made to exploit a specific targeting of Doxil ® to reduce the manifestation of this adverse reaction. This problem is of general significance, sincecutaneous reactions often lead to alterations of Doxil ®  dosing regimen in patients and mightsubsequently compromise the therapeutic outcome of cancer treatment. Tumor-bearing mice wereused to study the biodistribution and skin-tissue accumulation effects of the tumor-targeted Doxil ® (the clinically used anti-cancer formulation) coupled with the anti-cancer monoclonal 2C5 antibody(mAb 2C5) as well as AE caused by Doxil ®  application. The modification of Doxil ®  with mAb 2C5resulted in a significant decrease in the normal skin accumulation of doxorubicin compared to srcinalDoxil ®  and substantially reduced AE. The frequency of AE was decreased by 3-4-fold with the mAb2C5-modified doxorubicin-loaded long-circulating liposomes. Thus, targeting of Doxil ®  with theanticancer mAb 2C5 not only can increase the tumor-specific accumulation of the drug, but alsodiminishes the cutaneous side-effect of the srcinal Doxil ®  therapy. Keywords Doxorubicin; Long-circulating liposomes; Doxil ® ; Tumor targeting; Tumor-specific antibody; Anti-nucleosome antibody; Auricular Erythema INTRODUCTION The enhanced tumoral delivery of anti-cancer drugs in long-circulating pharmaceuticalnanocarriers is based on the passive accumulation of such carriers via the enhanced  permeability and retention (EPR) effect responsible for the extravasation of large moleculesand nanoparticles into the tumor mass in the cancerous areas with “leaky” vasculature (Iyer,et al. 2006). Doxil ®  [doxorubicin-loaded polyethylene glycol(PEG)-coated liposomes]embodies this principle of passive targeting. It was shown earlier, that PEG-liposomes *Corresponding author: Dr. Vladimir Torchilin, Department of Pharmaceutical Sciences, Northeastern University, Mugar Building,Room 312, 360 Huntington Avenue, Boston, MA 02115, USA; Tel: 617-373-3206; fax: 617-373-8886; e-mail: E-mail:v.torchilin@neu.edu. † Tamer Elbayoumi, PhD: Principal formulation research scientist, Atrium Medical Corp., 5 Wentowrth Dr., Hudson, NH, 03051 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.  NIH Public Access Author Manuscript  Int J Pharm . Author manuscript; available in PMC 2009 June 5. Published in final edited form as:  Int J Pharm . 2008 June 5; 357(1-2): 272–279. doi:10.1016/j.ijpharm.2008.01.041. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    demonstrate prolonged circulation in the blood (Klibanov, et al. 1990), and encapsulatingvarious drugs including doxorubicin within these sterically-stabilized PEG-liposomesfavorably alters their pharmacokinetics and biodistribution (Allen, et al. 1991,Papahadjopoulos, et al. 1991). This has resulted in the significant decrease in doxorubicin-associated dose-limiting toxicities: cardiomyopathy and myelosuppression (Berry, et al.1998, Safra, et al. 2000).However, due to this alteration of the pharmacokinetic profile of free doxorubicin, the dose-limiting toxicities of the long-circulating liposomal doxorubicin have been shifted towardsmucocutaneous reactions such as palmar-plantar erythrodysesthesia (PPE) and mucositis/stomatitis (Hamilton, et al. 2002, Lotem, et al. 2000, Uziely, et al. 1995). The current hypothesisfor the development of PPE is that the small size and long circulation time of Doxil ®  allow for the liposome accumulation in the skin where the basal layers of the skin are damaged with prolonged exposure to doxorubicin as the liposomes slowly release this drug with k nown vesicant properties. Moreover, liposomes with long circulation times were found to accumulatein the skin of experimental animals to a greater extent than liposomes with shorter circulationtimes (Allen and Hansen 1991, Papahadjopoulos, et al. 1991). This is believed to be a resultof the pressure-dependent extravasation of such li posomes into cutaneous tissues (Allen and Hansen 1991, Hamilton, et al. 2002, Hensley, et al. 2001), which is currently supported byexperimental and clinical data indicating that the accumulation of the long-circulatingliposomes in fact repeats the anatomical distribution of PPE lesions in regions of skin that aresubjected to the pressure or irritation, like the flexure creases of the hands, soles of the feet, or  belt lines (Gordon, et al. 2000, Hensley, et al. 2001, Lotem, et al. 2000). As indicated earlier,the incidence and severity of PPE associated with common Doxil ®  therapeutic regimens, byextrapolation of murine PK data to humans, can be controlled by increasing dose intervals,allowing time for the drug to clear  from skin and for the ulcers to heal. Nevertheless, the benefitof dose delay would be offset by reduced therapeutic efficacy (Charrois and Allen 2003,Ranson, et al. 1997, Uziely, et al. 1995).The modification of drug-loaded pharmaceutical carriers with tumor-specific ligands, such asanti-tumor monoclonal antibodies, is used to achieve higher local drug concentration at thetarget zone and enhanced anticancer effect of the chemotherapeutic agent (Noble, et al. 2004,Park, et al. 2004). The idea of adding monoclonal antibodies or their fragments to passivelytargeted long-circulating PEGylated drug carriers, including liposomes, may not only render  the whole formulation more tumor-specific, hence demonstrating a superior therapeuticeffectiveness (Torchilin 2005), but could also result in decreasing drug presence in non-targeted areas, thus reducing possible adverse effects of drugs in long-circulating carriers ontonormal areas of the body.Earlier, we have described the monoclonal antinuclear antibody 2C5 (mAb 2C5) with thenucleosome-restricted specificity and unique ability to selectively bind to the surface of a broad variety of lymphoid and non-lymphoid tumor cells of murine and human srcin (but not to thesurface of normal cells) via the tumor cell surface-bound nucleosomes released from the apoptotically dying neighboring tumor cells (Iakoubov, et al. 1995, Iakoubov and Torchilin1997). We have also used this antibody as a targeting moiety to actively target various drug-loaded long-circulating pharmaceutical nanocarriers, including doxorubicin-loaded PEGylated liposomes (Doxil ® ) to different tumors (Elbayoumi, et al. 2007, Elbayoumi and Torchilin2006, Elbayoumi and Torchilin 2007, Gupta, et al. 2005, Lukyanov, et al. 2004).Here, we report our findings from the experiments with animals receiving different Doxil ® formulations, plain and immuno-modified with the mAb 2C5. The animals subjected to srcinalDoxil ®  treatment exhibited a blistering skin reaction, limited to the auricular region of the ears,hence named “Auricular Erythema” (AE). The manifestations of this AE cutaneous reaction Elbayoumi and TorchilinPage 2  Int J Pharm . Author manuscript; available in PMC 2009 June 5. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    closely resemble the PPE noticed earlier known for doxorubicin in PEG-liposomes. Theoccurrence and severity of the AE can be correlated with the reported drug release from theliposomes and its skin accumulation profiles. The data also suggest that the modification of Doxil ®  with the tumor-specific mAb 2C5 significantly reduces the manifestation of AE.Therefore, mAb 2C5-modification of Doxil ®  can potentially help alleviate the therapy-associated mucocutaneous adverse effects known for the srcinal Doxil ® . MATERIALS AND METHODS Materials Doxil ®  was purchased from Pharmaceutics Inc. (West Roxbury, MA). Cholesterol (Chol), fullyhydrogenated soy phosphatidylcholine (HSPC), N-(carbonyl-methoxy poly (ethylene glycol2000)-1,2-distearoyl- sn -glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE),and phosphatidylethanolamine (PE) were from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Doxorubicin was purchased from Sigma Chem., Inc. (St.Louis, MO). Triethylamine (TEA), octyl glycoside (OG) and diethylene triamine pentaaceticacide anhydride (DTPA) were also products of Sigma. Poly-oxyethylene3400-bis(p-nitrophenyl carbonate) [PEG(pNP) 2 ] was purchased from SunBio (Orinda, CA). Cell culturemedia, DMEM, EMEM and RPMI 1640, fetal bovine serum (FBS) and concentrated solutionsof Na-pyruvate, non-essential amino acids, L-glutamine and penicillin/streptomycin stock solutions were purchased from CellGro (Kansas City, MO). Female BALB/C and C57/BLmice were purchased from Charles River Laboratories (Cambridge, MA). All solvents and other chemicals were analytical grade preparations.Monoclonal 2C5 antibody was produced in ascites via the I.P injection of 1.5×10 6  cells of thehybridoma cell line (obtained in our laboratory) into pristine primed BALB/C 4-week old malemice. The production and the purification of the mAb 2C5 were carried out by HarlanBioproducts (Indianapolis, IL). Methods  Antibody modification To prepare mAb 2C5 conjugates with PEG3400-PE, a 40 molar excess of p-nitrophenylcarbonyl(pNP)-PEG-PE prepared as in (Torchilin, et al. 2001), dispersed in 10 mg/ml solution of OG in 5 mM Na-citrate, 150 mM NaCl, pH 5.0, was added to an equal volumeof 16.7 μ M of the antibody in Tris-buffered saline (TBS), pH 8.5. The mixture was incubated for 24 hr at pH 8.5 at 4°C (Lukyanov, et al. 2004). Preparation of antibody-modified liposomes To modify doxorubicin-loaded PEG-liposomes, the mAb 2C5-PEG3400-PE conjugateobtained as described above was co-incubated with Doxil ®  preparation. For this purpose, thereaction mixture form the conjugation reaction (see the previous paragraph) was mixed in equalvolumes with Doxil ®  and incubated for 6-to-8 h at 4°C. Following the incubation, theremaining impurities and non-incorporated mAb 2C5 were removed by dialysis using celluloseester dialysis tubes with a cutoff size of 250,000 Da. The complete removal of the free antibodywas confirmed by the presence of only one peak of liposomes on the gel-chromatogram of thefinal sample (with no peaks corresponding to free mAb 2C5-PEG-PE or mAb 2C5-PEG-PEmicelles) (Elbayoumi and Torchilin 2007, Lukyanov, et al. 2004). Preparation of control liposomes Control PEGylated liposomes mimicking Doxil ®  composition but containing no doxorubicinwere prepared using the same lipid components and in the same concentrations as in Doxil ® . Elbayoumi and TorchilinPage 3  Int J Pharm . Author manuscript; available in PMC 2009 June 5. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    A lipid film was obtained from MPEG2000-DSPE (3.19 mg/ml), HSPC (9.58 mg/ml), and Chol (3.19 mg/ml). The lipid film was suspended in HEPES-buffered saline (HBS), pH 7.4,and sonicated with a probe-type sonicator at 10 Watts power for 15 min, followed by several passages through the mini-extruder with 100 nm pore size polycarbonate filter, untilapproximately 100 nm liposomes with narrow size distribution were obtained (Elbayoumi and Torchilin 2007, Lukyanov, et al. 2004). In vitro release of doxorubicin from liposomes The in vitro  release of doxorubicin from different Doxil ®  formulations was conducted inDMEM cell culture medium containing 10% fetal bovine serum (FBS). Li posomes at aconcentration of 0.5 mg/ml of doxorubicin, diluted in the media, were sealed into dialysis tubeswith the cutoff size of 12,000–14,000 Da. The liposome-loaded dialysis tubes were incubated in 50 ml of the media for 48 h at 37°C, with continuous stirring. At various time points, aliquotswere withdrawn, and replaced with the equal volume of the media. Doxorubicin concentrationswere measured at 485 nm using a Hitachi U-1500 spectrophotometer, Hitachi Instruments(Schaumburg, IL) (Ishida, et al. 2001, Xiong, et al. 2005). 111 In radiolabeling of liposomes Doxil ® -mimicking liposomes containing the amphiphilic chelate DTPA-PE(HSPC:Chol:MPEG200-DSPE:DTPA-PE in 3:2:0.3:0.3 molar ratio) were prepared along withthe mAb 2C5-immunoanalogues. The loading of the liposome-incorporated DTPA-PEwith 111 In was performed via the transchelation mechanism from a weak citrate complex.DTPA-PE-containing liposomes were supplemented with 0.1 M citrate buffer and incubated for 1 h with 111 In (as 111 In chloride in citrate buffer) at RT, and then dialyzed overnight againstHBS at 4°C to remove the free label (Elbayoumi and Torchilin 2006, Torchilin, et al. 2001). Growth of tumors in mice Murine breast carcinoma (4T1) and colon carcinoma (C26) tumors were implanted in 8 week-old BALB/C mice by the subcutaneous injection of 10 5  cancer cells into the fat pads in thelower abdominal region. Similarly, murine Lewis lung carcinoma (LLC) cells were S.C.injected in 8 week-old C57/BL mice (5×10 4  cells/mouse). The time for the appearance of the palpable tumor (varies from one cell line to another and usually is 7–12 days. Mice wereregularly monitored, with free access to food and water (following animal care protocol no.R01210 approved by Northeastern University Institutional Animal Care and Use Committee,in accordance with the “Principles of laboratory animal care”, NIH publication no. 85–23,revised in 1985), and tumor volumes were calculated by measuring the length and width of thetumor at regular intervals (Chakilam 2004, Elbayoumi and Torchilin 2006). Single-dose pharmacokinetics and biodistribution of 111 In-labeled liposomes  In vivo  biodistributon studies of 111 In-radiolabeled Doxil ® -mimicking liposomal formulationalong with mAb 2C5-modified analogue were performed in 8 week-old female BALB/C mice, both healthy and 4T1 tumor-bearing (tumor volume range: 200–300 mm 3 ), in two separateexperiments. In each experiment, BALB/C mice were injected with 0.1 ml of 1 mg/ml 111 In-radiolabeled Doxil ® -mimicking liposomes via the tail vein. At 15, 30, 120, 360, 720, 1440 minutes post injection, blood was collected using a Pasteur pipette from the retro-orbital plexusof the eye, and the mice were euthanized by CO 2  followed by collection of different organtissues. The amount of radioactivity was quantified as CPM using a Beckman 5500B gamma-counter, and the amount of the accumulated radioactivity per gram of tissue was calculated followed by calculation of the temporal biodistribution and tissue accumulation parameters(Chakilam 2004, Elbayoumi and Torchilin 2006, Pastorino, et al. 2003). Elbayoumi and TorchilinPage 4  Int J Pharm . Author manuscript; available in PMC 2009 June 5. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t     Assessment of auricular erythema as Drug-induced side effect Starting one week after the initiation of the drug administration (given in a sub-therapeuticdose regimen, as 2.2 mg/kg/dose every 5 days, for four consecutive doses) into tumor-bearingmice, location, count and severity of any developed auricular erythma (AE) lesions weremonitored and documented for the rest of the treatment, in BALB/C mice bearing C26 tumors,and C57/BL bearing LLC tumors. Finally, AE percentage (AE%) was estimated for each group. Statistics Differences in the apparent AE occurrence percentage, and percentage of ears exhibiting AEmanifestations during the treatment were compared using logistic regression, and comparingthe odds ratio (OR) between the treatment groups [(AE events vs. no event) of mAb-2C5-modified Doxil ®  intervention/(AE events vs. no event) of srcinal Doxil ®  intervention]. Thesignificance level for the OR proportion of probability (ORp) was set at 0.1. (Batist, et al.2006, Greenhalgh 1997, Potamianou, et al. 2005). RESULTS & DISCUSSION Doxorubicin leakage from antibody-modified liposomes Similar to earlier data (Elbayoumi and Torchilin 2006, Elbayoumi and Torchilin 2007,Lukyanov, et al. 2004), the post-insertional technique for immuno-modification of Doxil ®  withthe mAb 2C5-PEG-PE conjugate did not markedly alter the physicochemical properties of thesrcinal Doxil ®  formulation and resulted in the final preparation of 112±7 nm liposomes with73±3 antibody molecules attached per single liposome. The complete release profiles (Fig. 1, panel A) of the srcinal Doxil ®  and mAb 2C5-Doxil ®  also did not show any significantdifference between these formulations. These in vitro  release profiles have also demonstrated a slow first order release of doxorubicin from its carrier with just about 30% drug release after two days, which extrapolates to ca. 80 % of the encapsulated drug to be released after approximately 5 days. This corresponds well to the estimated in vitro  doxorubicin release half-time of 85 hrs for similar HSPC liposomes (Allen, et al. 2005). This noteworthy prolonged release profile of the drug from the liposomal carrier, extending for up to 5 or even more days,evidently plays an important part of sustaining an effective local tissue concentration at thetumor site, combined with an extended doxorubicin tumor half-life reaching up to 70 hrs, asdemonstrated earlier by other groups (Charrois and Allen 2003, Charrois and Allen 2004,Papahadjopoulos, et al. 1991). On the other hand, it may play a causative role in the necroticmanifestations at normal mucocutaneous tissues, especially on the multiple dosing treatmentregimen of Doxil ® . Especially when considering the reported protracted skin tissue half-life(approx. 80–110 hr) of the released drug (Charrois and Allen 2003, Charrois and Allen2004). In vivo  biodistribution and skin accumulation of 111 In-labeled liposomes Using liposomes mimicking the lipid composition of Doxil ®  and radiolabeled with 111 In, thetissue biodistribution of different liposomal formulations was determined. Blood clearancedata of analogous formulations in tumor-bearing BALB/C mice reported earlier (Elbayoumiand Torchilin 2006), indicated that mAb 2C5-Doxil ® -mimicking liposomes cleared faster fromthe body compared to plain liposomes (MRT ≈  12 hrs for mAb 2C5-liposomes, compared toappr ox. 17 hrs for plain liposomes, as derived from AUC data using a non-compartmental analysis model). This somewhat accelerated clearance of the antibody-modified li posomesfrom the body can be attributed to the presence of antibody molecules on the liposomal surface,which made liposomes more prone to the uptake by RES cells, and this is true for the caseswhen both tumor-specific and non-specific immunoglobulins are present on the PEG-liposomesurface (Chakilam 2004, Xiong, et al. 2005). However, mAb 2C5-immunoliposomes still Elbayoumi and TorchilinPage 5  Int J Pharm . Author manuscript; available in PMC 2009 June 5. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  
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