Review For reprint orders, please contact [email protected]

Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy While combination chemotherapy has led to measurable improvements in cancer treatment outcomes, its full potential remains to be realized. Nanoscale particles such as liposomes, nanoparticles and polymer micelles have been shown to increase delivery to the tumor site while bypassing many drug resistance mechanisms that limit the effectiveness of conventional therapies. Recent efforts in drug delivery have focused on coordinated, controlled delivery of multiple anticancer agents encapsulated within a single particle system. In this review, we analyze recent progress made in multidrug delivery in three main areas of interest: co-delivery of antineoplastic agents with drug sensitizers, sequential delivery via temporal release particles and simultaneous delivery of multiple agents. Future directions of the field, in light of recent advances with molecularly targeted agents, are suggested and discussed. Combination chemotherapy has been a cornerstone of cancer treatment for over 50 years. Since the first observations of improved efficacy of multiple drug regimens against childhood leukemias, chemotherapy for most cancers consists of administration of multiple agents either simultaneously or closely sequenced. The success of these combinations is thought to hinge largely on the biological complexity and redundancy of cancer cells; through administration of multiple agents, each with its own biochemical target and accompanying toxicities, cancer cells are subjected to a multipronged attack that is much more difficult to tolerate. Ideally, administration of multiple agents can lead to synergistic efficacy, wherein the observed efficacy is greater than the sum of the efficacies of the individual drugs. Most drug combinations, therefore, were discovered empirically, through clinicians’ intuition into the intracellular effects of certain drug classes, but also by clinicians’ desire to maintain acceptable systemic toxicity. Therefore, each drug within a combination is selected based upon avoiding overlapping toxicities with the other agents, thereby allowing dosing to the highest tolerable level of each agent. Outcomes for some cancers have improved with the advent of combination chemotherapy; however, most multidrug chemotherapeutic regimens do not lead to durable remissions. Combination chemotherapy remains limited by nonspecific toxicity to healthy tissues and by cellular mechanisms within tumors that promote resistance to a wide variety of drug classes (i.e., multidrug resistance [MDR]).

Particulate drug-delivery systems offer means to alleviate the limitations of conventional chemotherapy. Nanoscale particles such as liposomes, polymer nanoparticles, dendrimers and nanoemulsions generally increase drug circulation lifetime within the bloodstream while improving delivery of anticancer agents to cancerous tissues through the enhanced permeability and retention effect. Particulate drug carriers can radically alter biodistribution as the pharmacokinetics of the carrier strongly influences the sites of accumulation. Through careful formulation and adjustment of drug retention within the particle, exact drug ratios can be maintained within a carrier, allowing for ratiometric dosing and exposure of tumor cells to synergistic drug ratios. Alternatively, precise temporal control of tumor exposure to drug can be realized with delivery vehicles; when extended to multiple drugs, true sequential drug exposure at the tumor site can be achieved. Multidrug delivery through single particulate systems is not without some disadvantages. As will be discussed below, formulation of multiple drugs within a carrier presents significant challenges. Pharmaceutical development activities must also demonstrate safety and biocompatibility of novel carrier compositions. Limitations can also be found in the clinic; whether by design or not, each drug is present in a precise ratio within the carrier that cannot be altered by the clinician. This feature limits potential dose alteration of either agent on a case-by-case basis. It remains to be seen whether this limitation will hinder the

Barry D Liboiron* & Lawrence D Mayer

10.4155/TDE.13.149 © 2014 Future Science Ltd

Ther. Deliv. (2014) 5(2), 149–171

ISSN 2041-5990

Celator Pharmaceuticals Corp, 810–1140 W. Pender Street, Vancouver, BC, V6E 3G1, Canada *Author for correspondence: Tel.: +1 604 708 5858 E-mail: [email protected]

149

Review | Liboiron & Mayer Key Terms Ratiometric dosing:

Administration and in vivo maintenance of chemotherapy drug duos at specific drug:drug molar ratios.

Targeted therapeutics:

Antineoplastic agents designed for specific targeting of molecules needed for carcinogenesis and/or tumor growth.

BCL2: Family of proteins

responsible for regulating cell death (apoptosis).

NF-kB: Protein complex that controls DNA transcription, cytokine production and is a factor in cell survival.

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use and acceptance of multidrug carrier systems in the clinical setting. „„Formulation

challenges of multidrug delivery systems Drug delivery has steadily expanded over the past 30 years to the point that virtually every class of cancer chemotherapy drug has been successfully incorporated into a nanoscale particulate carrier, with several single agent carrier systems approved for human use (e.g., Abraxane® [paclitaxel], Doxil® [doxorubicin; DOX] and DaunoXome® [daunorubicin]) for various indications. The logical extension of these systems is to attempt to incorporate multiple drugs into a single carrier to improve upon the benefits realized by conventional combination chemotherapy. Formulation of multiple drugs within a carrier system, however, can be difficult. The physicochemical properties of drug A and drug B are rarely similar; key parameters such as ionization state, water solubility and aggregation behavior can present considerable challenges to the successful and stable incorporation of any agent into a carrier. Two main strategies are employed. The simplest involves development and formulation of discrete vehicles for each drug, followed by physical mixing of the two encapsulated agents at the desired dosage or ratio. While this strategy allows for easier optimization of each drug carrier system, the pharmacokinetics and biodistribution of each particle may not be correlated with each other, which may impact the observed efficacy. The second strategy is co-formulation, in which each agent is formulated within the same particle. While this approach assures that particle-dependent biodistribution and pharmacokinetic behavior in vivo will be similar, formulation of multidrug carriers increases in complexity. Since encapsulation of multiple agents is usually achieved sequentially, drug loading procedures for the second and subsequent agents must attain stable encapsulation of the desired agent while maintaining an acceptable concentration of the first drug within the particle. Many chemotherapy agents interact with the carrier and potentially other encapsulated agents, frequently producing concentration-dependent effects on the release profiles of themselves and the other agent(s). Lastly, coordination of release rates of multiple agents, either to achieve ratiometric dosing or realize a specific temporal release pattern, is made more difficult since subtle chemical or structural changes to the carrier disproportionally affect both agents. Ther. Deliv. (2014) 5(2)

„„Classes

of multidrug-delivery systems Reports of multidrug-delivery systems have steadily increased over the past 10 years as researchers devise means by which the challenges discussed above can be overcome. To date, however, the vast majority report preclinical efficacy of novel multidrug carriers. To our knowledge, only two dual-drug carrier systems have proceeded to clinical trials. The purpose of this review is to describe the progress in the development of nanoscale pharmaceutical carriers to optimize therapeutic effectiveness of combination chemotherapy. Three broad strategies have emerged: n Co-delivery of antineoplastic agents with drug sensitizers, either direct inhibitors or downregulators (e.g., siRNA) of MDR mechanisms; Multidrug formulations that preferentially release one agent more quickly than the second, leading to true sequential drug exposure at the tumor site;

n

Combinations of multiple antineoplastic agents within a single carrier, designed to harness synergistic activity between the active drugs.

n

Each concept is presented through discussion of selected formulations that illustrate novel solutions to formulation and/or drug delivery challenges. Invariably improvements in drug delivery lead to increased efficacy over conventional ‘cocktail’ administration methods. Discussion of the impact of these formulations on current research efforts will be presented with an eye to future developments to extend multidrug delivery to newer classes of agents, such as targeted therapeutics. Combinations of chemotherapeutics & drug sensitizers Multidrug resistance of tumor cells remains a major challenge to cancer chemotherapy. Development of cellular drug resistance relies on two main mechanisms, either the active export of chemotherapeutic agents from the cytoplasm or altering of signaling/apoptosis pathways that promote survival of the tumor cell. Drug efflux transporters, such as ATP-dependent P-glycoprotein (P-gp), are often overexpressed in tumor cells and have been shown to actively pump out a wide variety of chemotherapeutic agents with disparate structures and chemical properties [1–4]. Tumor cells may also possess prosurvival mutations such as upregulation of future science group

Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy BCL2

[5,6] and NF-kB that alter the apoptotic response and allow the cells to tolerate chemotherapeutic damage [7–10]. For the purposes of the following discussions, drug sensitizers are defined as agents that do not themselves independently elicit a cytotoxic response but are able to markedly increase the cytotoxic potency of anticancer drugs they are combined with. Particulate drug -delivery systems provide a means to limit and/or eliminate the effectiveness of MDR mechanisms. Nanoscale carriers can be imported into the cell via endocytic and/or receptor-mediated pathways, decreasing intracellular drug export by membrane-bound P-gp and other ATP-binding cassette proteins [11–13]. Hydrophobic drugs typically require a carrier system to achieve delivery to the target site. These benefits have led to the development of several multidrug systems designed to decrease the effects of MDR and augment the therapeutic effects of a cytotoxic payload co-formulated in the same particle. In this section we review multidrug particulate systems that encapsulate an antineoplastic agent with a second (and possibly third) agent (i.e., a sensitizer) that serves to augment activity through disabling of cellular responses to the first agent. Since sensitizers individually have modest or no discernable anticancer activity, they are almost exclusively administered as part of combination therapy. For the purposes of this review we classify combinations of antineoplastics and sensitizers into those that include small-molecule inhibitors of MDR-related protein pumps (e.g., P-gp, MDR1; direct inhibition agents) and those that utilize nucleotide-based anti-MDR regulators such as siRNA and antisense oligonucleotides (ASOs), which cause indirect MDR interference through gene silencing.

Combinations of antineoplastics & direct MDR inhibitors A conventional strategy for circumvention of Pgp-mediated drug resistance is pairing an antineoplastic agent with a chemosensitizer, defined here as a small-molecule inhibitor of tumor cell drug resistance factors, typically proteins. A summary of multidrug particulate combinations of these drugs with antineoplastic agents is given in Table 1. Verapamil (Ver), a calcium channel blocker, has been shown to reverse P-gp-dependent drug resistance and was the most commonly used MDR modulator in earlier carrier systems [14,15] but was limited in its utility due to cardiovascular and nervous system future science group

| Review

toxicity. Delivery in a particle, therefore, could help to decrease systemic toxicity through more specific delivery of the drug to the target site. An early example reported by Wang et al. formulated verapamil with DOX in PEGylateddistearoylphosphatitylethanolamine, egg phosphatidylcholine (EPC) and cholesterol (Chol) liposomes (DOX antiresistant stealth liposomes; DARSL) [16]. Leakage of both drugs was described as minimal (less than 35% in human plasma after 24 h incubation), however, the presence of Ver increased the DOX leakage rate when compared with singly-formulated liposomal DOX. Release of Ver appeared to be considerably slower than DOX from DARSL. However, the coformulated liposomes were nearly 1800-times more cytotoxic to MLLB2 prostate cancer cells than free DOX, and approximately 1.8-times more so than concomitant administrations of free DOX and free Ver. DARSLs were also slightly more effective than co-administrations of liposomal DOX and liposomal Ver. Interestingly, administrations of free DOX and liposomal Ver were less efficacious than co-administration of the free drugs, suggesting that intracellular trafficking of the drug pair may need to be similar to achieve the greatest effect. In this case, coformulation of the two agents was critical to the observed cytotoxicity. The DOX/Ver pair was also formulated into targeted particles, through the covalent attachment of transferrin to similar drug-loaded PEGylated-distearoylphosphatityle thanolamine:EPC:Chol liposomes [15]. Recent efforts have focused on second and third generation molecules such as curcumin (Cur), quercetin, d-a-tocopheryl PEG1000 succinate (TPGS) and tariquidar (Tar). These agents are generally more specific and therefore less toxic to healthy tissue. While more potent against a number of MDR-associated proteins than verapamil, the natural flavone quercetin virtually requires drug delivery to avoid near complete binding to serum proteins [17]. Song et al. [18] systematically investigated formulation conditions that led to optimal size, stability and drug loading efficiency of quercetin and vincristine in poly(lactic-co-glycolic acid) (PLGA) nanoparticles produced through a solvent emulsion method, based on earlier optimization of a similar formulation of verapamil and quercetin [19]. Coformulation of two agents with large differences in hydrophobicity required careful tuning of the copolymer composition (lactide:glycolide), organic solvent composition, aqueous solvent additives and pH, as well www.future-science.com

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Review | Liboiron & Mayer Table 1. Selected dual-drug particulate formulations of antineoplastic and chemosensitizing agents. Antineoplastic Sensitizer agent

Formulation composition

Comments

Doxorubicin

Verapamil Verapamil

Both drugs encapsulated at high efficiency. Doxorubicin release is faster than verapamil, which may not be optimal Transferrin conjugation conducted on drug-loaded liposomes. In vitro drug release similar to the untargeted liposomes described above

[16]

Doxorubicin

Vincristine

Quercetin

PEG–DSPE–EPC–Chol liposomes Transferrin-conjugated EPC–Chol– (PEG)DSPE liposomes PLGA nanoparticles

[18]

Vincristine

Quercetin

Systematic study of the effects of formulation parameters on physicochemical properties and encapsulation efficiency Drugs encapsulated at synergistic 2:1 vincristine:quercetin ratio. Ratio well maintained in vitro

Paclitaxel

Curcumin

Doxorubicin

Curcumin

Paclitaxel

Tariquidar

Doxorubicin

Mitomycin C

Doxorubicin

TPGS

Paclitaxel

Parthenolide, PEG–DSPE–TPGS TPGS polymer micelles

ESM–Chol– PEG(ceramide) liposomes Flaxseed oil nanoemulsion PLGA–PVA nanoparticles PLGA nanoparticles with PEG–PLA–biotin label PEG–stearate–soybean oil–F68 nanoparticles Porphyrin–PLA nanoparticles

Ref.

Synergy considered but combination indices indicate combination is likely to be additive Produced single and dual-drug-loaded nanoparticles. Curcumin released faster than doxorubicin but dual-drug doxorubicin uptake was double that of singly formulated doxorubicin Very slow release of paclitaxel by in vitro assay may lead to increased cellular uptake of intact particles through biotin functionalization Drugs formulated at 2:1 ratio of mitomycin C:doxorubicin. Drug ratio well maintained in vitro Light sensitive polymer can trigger release upon irradiation. TPGS coating on particles, although free TPGS was better at inhibiting P-gp than the nanoparticle version. Photoactivation appears synergistic TPGS used to stabilize paclitaxel and parthenolide. Drugs released sequentially. Therapeutic role of TPGS unclear

[15]

[20]

[23] [24]

[25]

[26] [29]

[28]

Chol: Cholesterol; DSPE: Distearoylphosphatidylethanolamine; EPC: Egg phosphatidylcholine; ESM: Egg sphingomyelin; P-gp: P-glycoprotein; PLA: Poly(L-lactide); PLGA: Poly(lactic-co-glycolic acid); PVA: Poly(vinyl alcohol); TPGS: d -a-tocopheryl PEG1000 succinate.

Key Term Median effect ana­lysis:

Method to quantify drug interactions across a range of effect levels, rather than dose levels.

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as organic:aqueous solvent ratio. Predictably, some factors showed opposing relationships in entrapment efficiencies for the two agents. Optimization of the competing factors, however, allowed the authors to produce a formulation that achieved similar controlled release of both agents over 24 h [18]. This combination was later explored for ratio-dependent activity [20]. A variety of ratios of vincristine and quercetin were encapsulated in egg sphingomyelin liposomes. Cytotoxicity studies indicated strongly augmented cytotoxic activity for a 2:1 of vincristine:quercetin over ratios of 4:1 and 1:2 or single agent liposomes. A number of approaches have been used to co-formulate Cur with a cytotoxic agent. Cur affects multiple MDR mechanisms from blocking NF-kB to downregulation of three major ABC transporters (P-gp, MRP-1 and ABCG2 [21,22]) Ganta and Amiji formulated Cur with paclitaxel (Pac) in flaxseed oil-in-water emulsions [23], while, more recently, Misra and Sahoo formulated DOX with Cur in PLGA nanoparticles [24]. In both studies, the co-formulated particles were superior in downregulation of P-gp Ther. Deliv. (2014) 5(2)

and inhibition of anti-apoptotic pathways over equivalent doses of the free drugs. Examination of drug synergy between Pac and Cur, however showed only a very modest synergy (combination index = 0.94, where a combination index = 1 represents an additive response). Sahoo and Misra expanded upon the earlier work with Pac and demonstrated significantly increased amounts of DOX delivered to the nucleus of K562 leukemia cells over singly formulated DOX nanoparticles and free drugs in solution. The improved DOX accumulation may be due, in part, to the novel drug release properties of the formulation. Despite inefficient encapsulation (46%), release of DOX was markedly slower than Cur, with less than 50% of the total encapsulated drug released after 7 days. Cur was efficiently encapsulated (86%), but exhibited burst release behavior, with 50% of the loaded drug found in the external buffer within a day. These results are counter-intuitive; while more hydrophilic, DOX was released more slowly from PLGA nanoparticles than Cur, potentially leading to sequential exposure of the two agents within the target cells. future science group

Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy PLGA nanoparticles were also used to simultaneously encapsulate Pac and third-generation P-gp inhibitor Tar [25]. In this case the drugrelease properties were similar, with slow sustained release of both agents to the external 0.5% Tween®-80/PBS buffer (t1/2Pac approximately 15 days, t1/2Tar approximately 9 days). In vitro cell studies, however showed no difference between the cytotoxicity of the dual formulated particle and free drugs in solution. This result was inconsistent with cellular drug accumulation studies, which showed a doubling of intracellular Pac from Pac-Tar nanoparticles compared with the free drugs in JC and NCI-ADR-Res cells. Increased cellular accumulation of Pac did correlate, however, with decreased tumor growth and improved survival of Balb/c mice-bearing JC tumors, particularly when the nanoparticles were functionalized with biotin for improved targeting of tumor cells. In this case, the slow release of the drugs from the nanoparticles would be an advantage, as it would allow for accumulation of the intact particles at the tumor site, and a greater exposure time versus the generally rapid clearance of free drugs. Mitomycin C (MMC) was incorporated with DOX into hybrid polymer lipid nanoparticles [26], following on from earlier work with another P-gp inhibitor, Elaridar [27]. Drug concentration within the particle was optimized through extensive iterative formulation changes of the multicomponent lipid-polymer system. The two drugs were loaded at a 2:1 MMC:DOX ratio, previously identified as a synergistic ratio through median effect ana­lysis. Both drugs were well-retained within the particle in vitro, in contrast to earlier work with Cur and Tar, as described earlier. Dual-drug-loaded PLNs demonstrated greater cytotoxicity against wild type and drug-resistant MDA-MB435/LCC6 cells as compared with singly formulated particles and a physical mixture of two single formulations at the same 2:1 MMC:DOX ratio. The dose required to induce 4 log cell kill was also 20–30-times less than that required for the same cytotoxicity with the free drugs, representing a tangible improvement of in vitro efficacy for this drug pair. A number of groups have attempted to coformulate PEGylated vitamin E succinate (d-a-tocopheryl PEG1000 succinate, TPGS), an amphipathic P-gp inhibitor [28,29]. Shieh et al. incorporated TPGS into photoactive four-armed porphyrin–polylactide nanoparticles (PPLA) capable of photochemical internalization, future science group

| Review

wherein light irradiation causes endosome/ lysosome membrane rupture and subsequent drug release from the particles intracellularly [29]. Through the addition of DOX to the formulation, the authors combined three treatment modalities: chemotherapy (DOX), photodynamic therapy (photochemical internalization) and MDR inhibition (TPGS). Particles were pre-made via a solvent evaporation process in the presence of DOX and subsequently coated with TPGS. Inhibition of P-gp activity was modest for both the unloaded PPLA nanoparticles and those coated with TPGS compared with free TPGS and verapamil, however, the level of inhibition was significantly greater than the control. Photodynamic therapy was found to promote translocation of DOX from the cytoplasm to the nucleus, but only for DOX from TPGS-coated PPLA nanoparticles. A second study used TPGS as a stabilizing agent in a dual-drug formulation of Pac and parthenolide in PEG2000-DSPE micelles [28]. TPGS served to stabilize the encapsulated Pac and Par, preventing nearly 75 and 30% loss of the drugs, respectively, after 24 h. „„Combinations

of antineoplastics & nucleotide-based sensitizers A complementary strategy to targeted blocking or interference of MDR-related proteins is gene silencing, either through the use of siRNA or ASOs. While these agents show promise as MDR modulators, nucleic acid-based drugs are rapidly degraded within the bloodstream and only poorly transfected into target cells. Cationic lipid and polymer particles have typically been the most successful for stable encapsulation of nucleotide-based drugs based on electrostatic interaction between the anionic drug and the cationic carrier, but are hampered by unacceptable toxicity of the carrier components [30–32]. Dual-drug formulations of gene silencing and antineoplastic agents are summarized in Table 2 . Below we describe several systems containing such combinations and examine the effects of drug release, particle composition and structure on efficacy. Nucleotide-based MDR inhibitors were first packaged with a traditional cytotoxic drug (DOX) and antisense oligonucleotides (anti-MRP1 and -BCL2, individually and combined), in combination with the cytotoxic agent. Anionic liposomes composed of EPC, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and Chol were used to encapsulate various combinations of three components. Co-deliver www.future-science.com

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Review | Liboiron & Mayer Table 2. Dual-drug formulations of cytotoxic and nucleic acid-based multidrug resistance modulators. Antineoplastic Sensitizer agent (target)

Formulation composition

Comments

Doxorubicin

ASO (MRP1, BCL2) ASO, siRNA (MRP1, BCL2)

EPC:DPPG anionic liposomes DOTAP cationic liposomes

Drug release dependent on rate of liposome degradation

Doxorubicin

siRNA (BCL2)

Paclitaxel

siRNA (P-gp) siRNA (P-gp)

Mesoporous silica nanoparticles modified with polyamidoamine dendrimers PLGA–PEI nanoparticles

Doxorubicin

Doxorubicin

Anionic liposome– polycation–DNA

Doxorubicin

siRNA (BCL2)

PEI–PCL polymer core ± FA–PEG–PGA coating

Doxorubicin

siRNA (ASCL1)

Herceptin

ASO (HER2/ neu)

Functionalized gold nanorods with pendant poly(aspartate) PMLA functionalized nanoparticles

Ref.

Included inhibitors of both pump and non-pump MDR mechanisms. Designed version of particle for pulmonary administration Formulation optimized for pulmonary delivery. Negligible doxorubicin release over 24 h. Reducing environment greatly augments release. Intracellular delivery suggests endocytotic pathway Sequential release of siRNA followed by paclitaxel, but tumor growth inhibition incomplete Replacement of cationic lipid with anionic DOPA required for stable doxorubicin encapsulation. Transfection of siRNA still sufficient to assist paclitaxel intracellular accumulation of single formulation Folate-targeting moiety coats polymer micelle through electrostatic interaction. Applied after drug loading, coating reduces intrinsic toxicity of cationic carrier but does not interfere with transfection of siRNA payload Direct covalent link to doxorubicin, siRNA passively associated with poly(aspartate) coating. Doxorubicin release slower than siRNA due to layered structure Early example of a targeted therapeutic co-formulated with a second agent. Dual-drug polymer more effective than single versions

[33] [34–36]

[37,38]

[39,40] [41,42]

[43–45]

[46]

[47,48]

ASCL1: Achaete-scute complex-like 1; ASO: Antisense oligonucleotide; DOTAP: 1,2-dioleoyl-3-trimethylammoniumpropane; DPPG: Dipalmitoylphosphatidylglycerol; EPC: Egg phosphatidylcholine; FA: Folic acid; MDR: Multidrug resistance; P-gp: P-glycoprotein; PCL: Poly(caprolactone); PEI: Poly(ethyleneimine); PGA: Poly(glutamic acid); PLGA: Poly(lactideglycolic acid); PMLA: Poly(malic acid).

of all three agents prevented upregulation of MRP1 and BCL2 protein expression, in contrast to carriers encapsulating only DOX [33]. Cationic liposomes have been applied to the delivery of siRNA and antineoplastic agents. Hydrophilic drugs can be stably encapsulated within the aqueous internal volume, surrounded by a positively charged membrane for electrostatic interaction with siRNA. Minko and co-workers encapsulated DOX in preformed 1,2-dioleoyl3-trimethylammonium-propane liposomes followed by binding of MRP1 and BCL2 siRNA at a +/- charge ratio of 4:1 for targeting of both pump (MRP1) and non-pump (BCL2) mechanisms of MDR [34]. While the formulated liposomes were large (~500 nm diameter), fluorescence labeling and microscopy of the individual formation components demonstrated cytosolic and nuclear association of all three drug components with lung cancer cells in vitro. Delivery of this combination of multiple target siRNA or ASOs and DOX has also been explored for inhalation treatment of lung cancer by pulmonary delivery, using both the cationic liposome vehicle described above [35,36] and also 154

Ther. Deliv. (2014) 5(2)

through the use of mesoporous silica nanoparticles (MSNs) [37,38]. For MSN delivery, direct conjugation of both the siRNA components and DOX or cisplatin was necessary to achieve a stable system, although loading efficiencies of the antineoplastic agents were 40% [37] or lower [38]. Maximal inhibition of MRP1 and BCL2 gene expression was achieved with a mixture of DOX- and cisplatin-containing MSNs, which in turn were labeled with either MRP1- or BCL2targeted siRNA. Direct local delivery via inhalation resulted in 73% of administered MSNs to localize in the lungs, compared with 5% when administered intravenously. While pulmonary delivery to tumors has the potential to limit systemic toxicity in the treatment of lung cancer, these particles take advantage of a specific administration modality that is not readily applied to a broader class of malignancies. Amphiphilic polymer micelles are finding increased application in the delivery of nucleotide-based agents through the use of positively charged polymer components. Patil et al. reported the encapsulation of Pac with P-gp targeted siRNA in PLGA-polyethylenediamine future science group

Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy (PEI; a cationic polymer) nanoparticles with greater than 80% efficiency for both agents. PEI significantly increased siRNA encapsulation, improved its release profile and gene silencing efficacy over formulations composed of PLGA alone [39,40]. In vitro release of siRNA was considerably faster than Pac, with half-lives of approximately 5 and 10 days, respectively, suggesting that tumor cells would be exposed to the gene silencing agent first, prior to Pac exposure. Presumably, anti-(P-gp) siRNA would need to be present as the cell responds to the chemotherapeutic challenge posed by Pac, not significantly before such exposure. Temporal release characteristics of this formulation were not optimized and complete inhibition of tumor growth was not achieved [40]. While generally superior for siRNA and ASO loading, cationic polymer and lipid particles have greater toxicity than negatively charged particles of similar size. Attempts to avoid or mitigate this toxicity have been reported for dual-drug carriers. Chen et al. developed a synthetic guanidium-containing cationic lipid (DSA A, N,N-distearyl-N-methyl-N-2-(N´arginyl)aminoethyl ammonium chloride) that was also a downregulator of mitogen-activated protein kinase signaling [41,42]. Co-formulation with siRNA produced a dual-drug particle in which the carrier itself was an active agent that could act synergistically with the siRNA payload [42]. Substitution of the cationic DSAA lipid with anionic 1,2-dioleoyl-sn-glycero3-phosphoethanolamine in the formulation improved subsequent encapsulation of DOX, at the expense of reduced siRNA transfection efficiency in vitro. Induction of apoptosis and tumor growth inhibition, however, were unaffected by the differences in the two formulations, indicating that the reduced efficiency of siRNA gene silencing did not compromise the in vivo performance of the anionic liposome system [41]. The addition of a PEG layer to shield the positive surface in vivo has been shown to decrease this toxicity but can interfere with oligonucleotide incorporation within the particle. Shuai and co-workers developed a hierarchical assembly strategy to co-encapsulate two agents within PEI–PEG-poly(caprolactone) (PCL) diblock copolymer micelles [43]. DOX and anti-BCL2 siRNA were sequentially encapsulated in PEIPCL polymer micelles, which were subsequently surface coated with folate-labeled polyanionic PEG-poly(glutamic acid) (FA–PEG–PGA) for future science group

| Review

improved targeting of cancer cells [44]. Systematic variation of the PEI–PCL polymer:siRNA and FA–PEG–PGA:PEI–PCL ratios allowed for identification of a particle composition that provided optimal drug retention stability, cell internalization and cytotoxicity of the PEI–PCL carrier, as monitored by particle size, surface charge and in vitro cell viability assays, as shown in Figure 1. Increased loading of anionic siRNA resulted in surface charge neutralization (Figure 1A) with the observed z potential steadily increasing as the siRNA proportion of the ratio decreased (i.e., higher N/P [PEI–PCL:siRNA ratio]). At N/P ratios of 20 or higher, particle size stabilized at approximately 120 nm. Cytotoxicity steadily increased with higher N/P ratios (Figure  1C , circles). From these results, the authors selected an N/P ratio of 30 as an optimal compromise between surface charge, particle diameter and cytotoxicity of the polymer carrier. The effect of FA–PEG–PGA coating on system properties (Figure  1B) was then analyzed in a similar fashion. Physicochemical properties were largely unaffected by FA–PEG–PGA ratios (C/N) of less than 1/20 other than an expected decrease in surface charge. Higher C/N ratios (>1/10) caused particle instability as observed by increased particle size. Cytotoxicity decreased with increased FA label concentration with nearly 100% cell viability for the FA–PEG– PGA-coated particle versus approximately 65% for the unlabeled system at a P/N of 30. This meticulous characterization of physicochemical properties produced a dual-drug particulate system with augmented DOX activity against several cell lines with effective blocking of BCL2 anti-apoptosis mechanisms [43–45]. Gong and co-workers constructed multifunctional gold nanorods for simultaneous delivery of neuroendocrine-targeted siRNA and DOX [46]. Agent-specific linkages were used for binding to the nanorod through thioglycolates covalently linked to the gold surface through Au-S bonds. DOX was conjugated through hydrolysable hydrazone linkages while siRNA was associated electrostatically to cationic poly-l-arginine chains bound to the functionalized nanorod. DOX release was highly pH-dependent, with less than 15% over 60 h at pH 7.4, compared with greater than 90% at pH 5.3. This feature was promoted as a means to reduce systemic toxicity and increase delivery to the tumor site since release at physiological pH would be minimal while potentially enabling drug cleavage upon www.future-science.com

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Review | Liboiron & Mayer

30

100

20

10

50

Particle size (nm)

150

1400

ζ potential Size

20 450

10

300 0

0

N/P Ratio 20

10

30

1/2.5

1/5

1/10

1/20

N/P ratio

-10 1/40

0

D–PCE/BCL-2

40

D–PCE

30

B–PCE

20

B–PCE

10

1/80

0 5

30

1200

150 0

40

ζ potential (mV)

40

ζ potential Size

ζ potential (mV)

Particle size (nm)

200

C/N ratio

40

100

60 40

1/2.5

1/5

1/10

1/20

B–PCE/SCR

0

B-PCE/SCR/FA B-PCE/SCR 1/40

20

1/80

Cell viability (%)

80

C/N ratio

Figure 1. Formulation optimization of a dual-drug carrier through physicochemical and biological characterization. Dependence of PEI–PCL polymer micelle physicochemical properties with (A) polymer to siRNA ratio and (B) FA–PEG–PGA coating to polymer ratio. (C) Cytotoxicity properties of scrambled siRNA-loaded PEI–PCL particles with and without FA–PEG–PGA coating. B–PCE: Unloaded PEI–PCL blank particle; B–PCE/SCR: Blank PEI–PCL particles loaded with SCR; B–PCE/SCR/FA: Blank PEI–PCL particles loaded with SCR and FA; C/N: FA–PEG–PGA:PEI–PGA ratio; D–PCE: Doxorubicin-loaded PEI–PCL particle; D–PCE/BCL-2: Dual-loaded DOX/siRNA PEI–PCL particle; FA: Folic acid; N/P: PEI–PCL:siRNA ratio; PCL: Poly(caprolactone); PEI: Polyethylenimine; PGA: Poly(glutamic acid); SCR: Scrambled siRNA. Reproduced with permission from [44] .

cellular internalization within acidic endosomes. The siRNA component, however, was the most cytotoxic component of the system with the dual-drug system exhibiting only a minor decrease in viability of BON carcinoid cells while inclusion of a targeting moiety (octreotide) in the formulation provided a modest augmentation of each active component, particularly after drug incubation times were increased beyond 24 h [46]. 156

Ther. Deliv. (2014) 5(2)

More recently, Penichet, Ljubimova and coworkers reported two systems with covalently attached anti-tranferrin receptor or the immunostimulatory cytokine IL-2 (fused with antiHER2/neu antibody for targeting of HER2/ neu positive breast cancer cells, anti-HER2/ neu) covalently linked, along with ASOs, to a polymalic acid backbone for simultaneous targeting/immunostimulation and antiangiogenesis [47,48]. A schematic representation of the future science group

Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy

Dual-drug formulations with temporal release properties The rate of drug release from a carrier can have a dramatic effect on the observed efficacy in vivo [49]. Particulate carriers require a period for accumulation at the target site, delivery into cells and ultimately exertion of their antineoplastic effects. In some cases, controlled sequential release in which one agent follows the other over a certain period of time is preferred, as could be envisioned for some combinations of sensitizers and antineoplastic agents described in the previous section. Several dual-drug delivery systems exhibited much greater intracellular drug concentrations and consequent cytotoxicity due to previous inhibition of drug export proteins, over and above that which could be achieved by single formulations of either agent. In other cases, simultaneous disruption of multiple cellular pathways is desirable to overwhelm cellular responses to chemotherapy challenges and induce cell death. The reliance of combination therapy on these principles requires meticulous formulation design and iteration to ensure the drug-release properties of a multidrug formulation are optimal for the combination of interest. Achieving drug synergy in vivo is predicated upon exposing the target site to sufficient concentrations of each agent but also at the most appropriate sequence on a timeline. Unfortunately, many multidrug particulate systems were designed on an ad hoc basis, in that the release properties of one or both drugs were simply measured (or even ignored) and viewed as a drug property and not a key characteristic to be optimized. Recently, greater attention has been devoted to the importance of drug release and retention, and some systems have been designed future science group

–– –

–

–

––

– –

–– –– –

–

– – – ––

–

– – –– – S

–

–

–– –– –

–

– – – –– – – –– – S

S

S

Polymalic acid/leucine ethyl ester copolymer S

S S –– –

–

–

S –– – – – – – – –– – – – – –– – – – ––

–

–– –– –

–

–

– – – –

– – –– –

AON α4

mPEG5000

PEG3400

IL-2 bioconjugate is shown in Figure 2 [48]. The ASOs were covalently linked to the polymer backbone via a biodegradable disulfide linkage, while the fusion protein IL-2:anti-HER2/neu was attached to a pendant PEG3400 to attempt to maintain activity of the protein conjugate. The particles, approximately 27 nm in diameter, were found to stimulate an immune response in vivo as well as significantly improve survival of mice bearing murine syngeneic mammary tumors over the IL-2:anti-HER2/neu fusion protein alone. While the above reports represent a new conceptual platform, multirole particles with specific targeting moieties and biologic components are likely to represent the next phase of multiple drug delivery.

| Review

Fluorophore

–

AON β1

Anti-HER2/neu IgG3-(IL-2) fusion

Figure 2. Schematic representation of a multicomponent polymalic acid-based nanoparticle bioconjugate system. AON: Antisense oligonucleotide. Reproduced with permission from [48] .

with a means to carefully tune the drug release rates. In the following sections, we discuss the challenges and progress made to construct multidrug systems with rationally designed temporal drug release. We first examine some examples of systems with true sequential release of each agent while the second section will discuss multidrug carriers in which drug-release rates are matched, allowing for delivery of specific drug ratios. In both cases, these sections provide examples of formulations in which drug release is viewed as a system property, in which the physicochemical characteristics of the active agents, carrier matrix and other excipients are considered in concert to maximize therapeutic benefit. „„Sequential

delivery of multiple agents The ability to both radically alter, yet finely tune the pharmacokinetics and/or biodistribution of encapsulated anticancer agents allows for the development of true sequential delivery vehicles for multiple drugs. While anticancer agents can be sequenced in an elementary way simply through timing of dose administration in the clinic (as can drug carrier systems, e.g., with sequential injections of singly formulated liposomal DOX and combretastatin A4 alternating every 2 days [50]) these methods frequently require multiple administrations over an extended period. Through careful, iterative www.future-science.com

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Review | Liboiron & Mayer formulation, particulate delivery systems can be tuned to sequentially release multiple agents, often in the vicinity of, if not within, the tumor cells. This section will examine reported multidrug delivery agents that were rationally designed to sequentially release each agent to achieve a superior therapeutic effect over and above conventional sequential administration of each agent. These formulations (listed in Table  3) distinguish themselves from serendipitous sequential release of multiple agents through deliberate modifications, derivatization chemistry or carrier manipulations that allow for true sequential exposure of each agent over a defined time period. Combinations of antineoplastic drugs with anti-angiogenesis agents offer the potential to trap a cytotoxic payload within the tumor following degradation or ‘normalization’ of the tumor vasculature. Careful tuning of release rates would be required for such a system to avoid premature reduction of tumor blood vessel permeability prior to sufficient accumulation of the encapsulated cytotoxic agent. Despite this difficulty, several combinations of cytotoxic drugs with combretastatin, an anti-angiogenesis agent, have been reported. In a landmark paper, Sengupta et al. reported a dual-drug formulation of DOX and combretastatin [51]. DOX was covalently tethered to a PLGA5050 polymer chain via a hydrolysable amide linkage, with subsequent nanoparticle formation through an emulsion/solvent evaporation technique. While this procedure produced nanoparticles with a higher polydispersity, the authors were able to select desired size fractions through ultracentrifugation and extrusion through 100 nm membranes. Nanoparticles of approximately 110 nm diameter were then coated with a phospholipid (PEG 2000 –DSPE:DSPC:Chol) membrane in

the presence of combretastatin, to form a dualdrug ‘nanocell’ with an average diameter of approximately 180 nm. Due to its hydrophobicity, combretastatin was readily incorporated into the phospholipid coating. In vitro release of combretastatin was rapid, with virtually all drug released within 30 h while DOX release was slower, reaching approximately 50% at 30 h but requiring over 90 h for full release. Slower release of DOX was attributed to a step-wise degradation of the DOX–PLGA oligomer, which tended to fragment first, followed by hydrolytic cleavage of the parent drug. In vivo efficacy of the dual-drug-loaded nanocell was superior to singly formulated liposomes (combretastatin) and nanocells (DOX) when administered to B16/F10 melanoma or Lewis lung carcinoma-bearing mice. Tumor growth inhibition was greatest with the DOX/ combretastatin nanocell and was attributed to observed degradation of the tumor vasculature shortly after accumulation of the nanocell in the tumor, trapping the cytotoxic payload within. These effects were not observed with a dualdrug-loaded liposomal version, which was not expected to exhibit controlled temporal release of the two agents. Targeted delivery of this combination was reported by Zhang and co-workers who developed an arginine–glycine–aspartate (RGD)labeled liposome co-encapsulating DOX and combretastatin [52]. In vitro release of combretastatin, however, was very rapid in vitro with 50% released within 4 h in physiological saline, while DOX release remained under 20% for the duration of the assay (48 h). The targeting moiety improved the intracellular accumulation of DOX as measured by confocal microscopy, however, the higher levels did not correlate to a significant improvement in tumor growth

Table 3. Dual-drug particulate formulations with designed sequential release characteristics. Drug 1

Drug 2

Combretastatin A4 Doxorubicin

Combretastatin A4 Paclitaxel

Gentamycin

Indomethacin; itraconazole

Formulation composition

Comments

Drug-conjugated PLGA core with phospholipid coating (‘Nanocell’) PLGA nanoparticles

Doxorubicin was conjugated to the internal polymer core while combretastatin was passively associated with the phospholipid layer to achieve sequential release Drugs loaded at unsupported 1:4 ratio. Sequential release achieved despite only passive paclitaxel encapsulation. Some evidence that combretastatin release assisted nanoparticle accumulation Release of each agent was well resolved. Delay before release of second agent was dependent on relative amounts of each drug carrier

Titania nanotube arrays containing drug-carrying micelles

Ref. [51]

[53,54]

PLGA: Poly(lactic-co-glycolic acid).

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[55]

Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy inhibition in mice bearing B16F10 tumors. The co-formulated, RGD-targeted liposomes exhibited only a modest improvement over untargeted dual-drug liposomes, which in turn was not significantly superior to single agent liposomal combretastatin. This result suggests that the majority of chemotherapeutic effect is due to the antiangiogenesis agent. Efficacy results did not include administration of free agents either alone or in tandem, which precludes elucidation of this result [52]. Other formulations have combined Pac with combretastatin [53,54]. The greater hydrophobicity of Pac over DOX allowed for co-formulation of the two agents within a smaller (~70 nm), homogeneous mPEG–PLA nanocapsules. Polymer ligation was again utilized to control release of Pac, while combretastatin was passively incorporated into the particle during self-assembly. This procedure produced a nanocapsule system with an average size considerably less than that of the nanocell and a more straightforward production method. In vitro combretastatin release remained faster than that of Pac, with a portion of the drug (~20%) remaining within the particle. Release of combretastatin appears to be slower than release of the drug from the nanocell formulation. These observations may reflect a greater stability of combretastatin within the nanoprecipitated mPEG–PLA particle versus passive encapsulation in a phospholipid coating. The dual-drug nanocapsule system, however, remained active in vivo against Lewis lung carcinomas. Subsequent work from Ho and coworkers reported a nontethered (i.e., passively encapsulated) Pac–combretastatin nanoparticle comprised of PLGA polymer, functionalized with RGD peptide for improved targeting to tumor and endothelial cells [53]. A new approach for true sequential release from a drug carrier utilized mechanical layering of therapeutic agents within titania nanotube arrays for local delivery to tumors. Losic and co-workers encapsulated hydrophobic and hydrophilic drugs within two types of polymer micelles layered within nanotubes [55]. This approach allowed for true sequential release first of the hydrophobic drugs (e.g., indomethacin and itraconzaole) layered at the top of the titania construct, followed by the encapsulated hydrophilic drug (e.g., gentamicin), well resolved temporally from the first agent. Release of the second drug was strongly dependent on the amount of the encapsulated first agent layered on top within the tube. future science group

| Review

„„Simultaneous

delivery of multiple agents: harnessing drug synergy in vivo As our understanding of cellular processes and responses to chemotherapeutic challenges improves, it is becoming increasingly clear that multiple agents do not act independently within the tumor cell. Significant interaction may take place between affected cellular pathways which may be the source of improvements in the observed efficacy. This concept is not surprising given the known signaling and communication pathways, redundancies and feedback loops associated with certain protein regulators within the cell [56]. Multiple disruptions of these pathways through simultaneous exposure to multiple agents can defeat or circumvent cellular defenses and lead to cell death. Achieving this result in vivo requires tumor cells to be exposed to the agents within a narrow period of time and at efficacious concentrations. Drug delivery of multiple antineoplastic agents through particulate carriers can achieve this goal, although the formulation challenges are considerable. Multiple agents, frequently with disparate physicochemical properties, must be encapsulated in a discrete carrier at sufficient concentrations (i.e., IC90 or greater), and released from the carrier at such a rate such that sufficient amounts of the active agents arrive at the target site. Conversely, an overly stable formulation decreases the bioavailability of the active agents so one cannot permanently ‘lock-in’ agents to the carrier to ensure delivery to targets. An early example of a dual-drug particulate carrier illustrates the difficulties encountered in formulation of such a system. Two antineoplastic agents, 6-mercaptopurine and daunorubicin, were encapsulated within neutral or anionic liposomes composed of phosphatidylcholine, Chol and cardiolipin [57]. Encapsulation of the two agents was markedly different. Daunorubicin was actively loaded with the liposome via a pH-gradient method with satisfactory efficiency (~60%); however, 6-mercaptopurine was passively loaded during liposome formation, with correspondingly poor efficiency of less than 2% into neutral and anionic liposomes. Furthermore, pre-encapsulation of 6-mercaptopurine strongly affected the efficiency of subsequent daunorubicin loading. To be a clinically viable formulation, the encapsulation efficiency of 6-mercaptopurine would have to be improved to yield a carrier system with sufficient efficacious amounts of both agents. Recent efforts to produce dual antineoplastic drug carrier systems are listed in Table 4. Below we highlight reports www.future-science.com

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Review | Liboiron & Mayer Table 4. Selected drug-delivery systems of multiple antineoplastic agents with methods of drug retention. Drug 1

Drug 2

Formulation type and composition

Comments

Ref

Passive encapsulation DOX

Paclitaxel Etoposide

Paclitaxel

Tanespimycin (17-AAG)

C6 -ceramide Rapamycin Topotecan Vincristine

DOX

Docetaxel

Developed separate particle for each agent. Paclitaxel (in LE) and DOX (in GEG) had similar retention in separate vehicles. Higher drug loading improved drug retention PEG–PLA polymer Entrapment of 17-AAG improved retention of paclitaxel over singly micelles formulated micelle. Retention half-life of either agent less than 4 h PEG–PLA or PEG– Release half-life of 17-AAG was half that observed for paclitaxel in DSPE–TPGS micelles buffer or serum PEG–PCL nanoparticles Zeta potential reduced upon ceramide encapsulation suggesting particle surface binding while paclitaxel occupies particle core GMO-coated iron oxide Both drugs partitioned to GMO coating, but release of rapamycin nanoparticles much faster (50% at 5 days vs 11 days for paclitaxel) Phospholipon 100H– Vincristine release slow and zero order while topotecan release Chol– (PEG–DSPE) somewhat faster. Presence of either drug has no effect on the other’s liposomes release rate (PEG) –PLGA with RNA DOX intercalated into RNA aptamer coating; docetaxel occupies aptamer coating particle core. DOX release appreciably faster

[58]

GEG or LE micelles

[59] [60,61] [62] [63] [64,65]

[66]

Single conjugation to carrier Cisplatin

Docetaxel

Cisplatin

DOX

[67]

(PEG) –PLGA

Pt(IV) prodrug conjugated to PLA chain. Successful use of tethering to coordinate release to two highly disparate agents Lysine-conjugated DOX pre-encapsulated in liposome, platinum prodrug conjugated via poly(acrylic acid) coated lysine to poly(acrylic acid) coating DPPC–DOPG liposomes

[69]

Double conjugation to carrier DOX

Aminoglutethimide HPMA copolymer–drug Drugs conjugated to HPMA backbone via enzyme-degradable conjugate linkage. Slower drug release associated with decreased efficacy; accessibility of cleavage site key to activity DOX Gemcitabine HPMA copolyme–drug Release rate of gemcitabine much faster than DOX, possibly due to conjugate steric accessibility of the drug conjugates. Release of gemcitabine slower for dual formulation suggesting steric effect of DOX DOX Wortmannin PEG–poly(aspartate Covalent linking of both drugs to block copolymer allows precise ratio hydrazide) control of multiple drugs, however hydrolysis rate may vary between different agents Cytarabine 5-fluorouracil Polyvinyl polymer Drugs conjugated to monomer vinyl groups, then co-polymerized to nanoparticles with produce random copolymer. Scant 5-fluorouracil release over 50 h at covalent pendant drugs pH 7.4. Very high drug:polymer ratio (~29% w/w) DOX Camptothecin PC– (PEG–DSPE) – (PLA– Drug–PLA conjugates form nanoparticle core surrounded by drug) lipid-coated PEGlylated lipid layer. Physiochemical properties of drug conjugates nanoparticles determined by PLA chain

[70,71]

[72]

[73]

[74]

[75]

Chemical degradation of carrier DOX

Paclitaxel

PEG–PLA polymersomes

DOX

Paclitaxel

PEG–PLA, PEG– polycarbonate polymersomes

DOX

Paclitaxel

PVA-coated iron oxide nanocapsules

Acid-labile polymersomes degrade through PLA end-hydrolysis leading to physical changes in particle shape. Pore formation leads to slightly faster DOX release over paclitaxel Degradable polysome showed faster paclitaxel release than previous example, but still slower than DOX. Possible polymer micelle formation during degradation leads to micelle formation and a reservoir for paclitaxel Acid sensitive release properties well tuned. Negligible drug release at pH 7.4, greatly accelerated in acid conditions. Release rate of paclitaxel sensitive to initial PVA concentration during formulation

[76]

[77]

[78]

AAG: Allylamino-17-demethoxygeldanamycin; Chol: Cholesterol; DOPG: Dioleolyphosphatidylglycerol; DOX: Doxorubicin; DPPC: Dipalmitoylphosphatidylcholine; DSPE: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; GEG: Poly(g-benzyl l-glutamate)/poly(ethylene oxide); GMO: Glycerol monooleate; HPMA: N-(2-hydroxypropyl)methacrylamide; LE: Poly( l-lactide)/poly(ethylene glycol); PCL: Poly(caprolactone); PLA: Poly(L-lactide); PLGA: Poly(lactic-co-glycolic acid); PVA: Poly(vinyl alcohol); TPGS: d -α-tocopheryl poly(ethylene glycol) 1000 succinate. 

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Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy in which multiple agents were delivered to the target site by a single carrier, at concentrations in which both drugs make measurable contributions to the observed in vitro and/or in vivo efficacy. The simplest approach for multiple drug delivery from a formulation perspective is to encapsulate each agent into a separate carrier system, followed by optimization of one or both systems to either normalize drug release properties or improve drug encapsulation efficiency. This approach grants considerable flexibility in adjusting individual carrier composition, encapsulation conditions and other formulation variants. Na et al. encapsulated DOX or etoposide in PEGylated poly(g-benzyl-l-glutamate) and Pac in PEGylated poly(l-lactide) polymer micelles and studied the in vitro cytotoxicity and in vivo efficacy of the encapsulated agents as single agents and given in combination [58]. Isobologram ana­lysis indicated possible synergistic cytotoxicity between DOX and Pac or etoposide. This approach offers several advantages in that each drug can be dosed up to its maximum tolerated dose, or at specific drug:drug ratios. Treatment of CT-26 murine tumorbearing mice with the dual-drug system yielded significantly improved survivability over the single agents given at equivalent doses. While the use of two carriers in this system provided some improvement over treatment with free drugs, further work would be required, such as biodistribution studies, to discern whether the in vivo performance is optimal. Disadvantages of the dual carrier design, aside from the obvious effort involved in optimization of two carriers, include introducing differences in delivery to the target site or uptake within cell due to the two disparate carriers, as well as a more complicated regulatory landscape. For these reasons, dual formulation within a single carrier is more common to ensure that differences in particle behavior do not detract from the observed efficacy. Several methods have been applied to encapsulate multiple antineoplastic agents in a single carrier. Passive encapsulation, in which the agents are reversibly associated with the carrier via equilibria processes, remains a common process and is most frequently achieved through generation of nanoscale carriers in the presence of the active agents. This method has been successfully applied to a variety of drugs in combination with Pac, such as tanespimycin (17-allylamino-17-demethoxygeldanamycin, or 17-AAG) [59–61], ceramide [62], and rapamycin [63], as well future science group

as the combination of topotecan and vincristine [64,65]. Most passively encapsulated combinations comprise agents with strong hydrophobicity to ensure stable association of the drugs with the forming hydrophobic core during particle generation. Water-soluble drugs, however, tend to remain in the aqueous phase or be loosely associated with the hydrophilic corona. Coordination of drug release rates, however, is difficult; release rates are dictated by the physicochemical properties of the drugs and the interactions with the carrier matrix. Efforts to improve retention of one drug can be detrimental to the retention of the second agent. Zhang et al. reported a formulation that provided different environments for DOX and docetaxel by design [66]. DOX was first associated to an RNA aptamer targeted to prostate specific membrane antigen, taking advantage of the well known nucleic acid intercalation properties of anthracyclines. Docetaxel was encapsulated in PEG-PLGA nanoparticles through a precipitation method, which were subsequently functionalized with the DOX-labeled RNA aptamers. While DOX release was substantially faster than docetaxel (50% released in less than 5 h vs approximately 7.5 h, respectively), fluorescence studies demonstrated that significant amounts of both agents were found within prostate-specific membrane antigen-positive cells, indicating that differences in drug retention may be at least partially overcome through the use of targeting ligands [66]. Emerging strategies for the encapsulation of chemically disparate agents involve covalent binding of one or more of the agents to either the carrier matrix or other moieties to alter the physicochemical properties of an agent(s). Kolishetti et al. formulated docetaxel and a Pt(IV) prodrug into PEG–PLGA nanoparticles [67]. Despite the wide differences in physicochemical properties, co-formulation of the two drugs was achieved through covalent linking of a Pt(IV) prodrug to a hydroxyl group of a PLA polymer derivative. This work follows on from an earlier report that reversibly linked Pac with nonpolar ‘anchors’ such as Chol and docosanol prior to encapsulation in PEG–polystyrene nanoparticles to achieve controlled rates of Pac release [68]. Kolishetti et al. demonstrated that this concept can be extended to hydrophilic drugs, such as cisplatin prodrugs. The degradable linkages to a long polymer chain allow for stable loading of the Pt(IV) prodrug into polymer nanoparticles. In vitro release of platinum was slower than the passively encapsulated www.future-science.com

| Review

Key Term Isobologram: Graphical

method to analyze for synergistic drug activity between two agents that indicates drug:drug concentrations at which maximum synergy is observed, but not necessarily maximum efficacy.

161

Review | Liboiron & Mayer docetaxel demonstrating the potential of covalent tethering of drugs to radically change their retention within a particle. Varying the length and composition of the hydrophobic PLA anchor to Pt(IV) could be used to match rate of Pt release to that of docetaxel. A comparable formulation was reported by Lee, O’Halloran and Nguyen, who encapsulated DOX in a liposomal core coated with a pH-responsive, cisplatin-conjugated polymer shell (termed ‘nanobins’) [69]. Similar to the previous example, the nontethered DOX was released from the liposome core considerably faster than cisplatin, with 50% released within 12 h compared with approximately 36 h for cisplatin. Synergism between the two agents was carefully studied through determination of combination indices at various drug effect levels and isobologram ana­lysis. The dual agent polymer-caged nanobins exhibited stronger synergistic cytotoxicity over combinations of both free and encapsulated single agents, possibly due to differences in cellular uptake of DOX. This result validates the general superiority of single carrier design for multiple agent delivery, particularly when attempting to observe synergistic activity. Other systems have tethered both drugs to the polymer backbone. Duncan and co-workers produced a library of conjugates containing the aromatase inhibitor aminoglutethimide and/or DOX tethered via hydrolysable peptide linkages to N-(2-hydroxypropyl)methacrylamide copolymer [70,71]. Conjugation of both drugs to the polymer chain had little effect on the particle. Release of the agents, as observed through in vitro enzyme-mediated hydrolysis assays, was slow with approximately 20% of each agent released after 5 h and the design of the system offers few opportunities to modify the drug release characteristics. Similar results were also observed by Lammers et al. in a system of DOX and gemcitabine again covalently attached to a N-(2-hydroxypropyl) methacrylamide polymer backbone [72]. Release of the agents was substantially different with greater than 75% of the conjugated gemcitabine released in less than 5 h, compared with less than 10% for DOX. Differences in drug release between the two agents may have been related to steric inaccessibility of the DOX linkage compared with gemcitabine, and the relative disparity in the size of the two agents. Kwon and co-workers linked DOX and the phosphotidylinositol-3 kinase inhibitor wortmannin to PEG–poly(aspartate hydrazide) block copolymers via hydrolysable hydrozone 162

Ther. Deliv. (2014) 5(2)

bonds [73]. Both singly formulated and dual formulation versions of the polymer chains were synthesized, allowing for examination of the differences in physicochemical and biological properties of chemically mixed micelles (both drugs on the same polymer chain) and physically mixed micelles (each polymer chain is conjugated homogeneously to one drug). In both cases, while formulated drug ratios could be precisely controlled, hydrolysis rates of either drug from the polymer chain were not reported. Cytotoxicity of each agent against MCF-7 cells was approximately tenfold less toxic with no significant differences between chemically mixed micelles and physically mixed micelles composed of 1:1 DOX:wortmannin but possible synergistic cytotoxicity between the two drugs at that ratio. Based on these results, mixed polymer micelles of multiple drugs could be prepared through simple mixing of appropriate amounts of singly conjugated polymer chains of each agent. The dual conjugation strategy has also been used to deliver combinations of cytarabine and fluorodeoxyuridine [74], as well as physically mixed lipid coated nanoparticles of DOX–PLA and camptothecin–PLA conjugates [75]. Chemical breakdown of the particle matrix is a common strategy to induce drug release at the target site. Several groups have used pHsensitive polymers to construct liposome-like vesicles (termed ‘polymersomes’) for drug delivery. A polymersome composed of PLA and PCL was used to co-encapsulate Pac and DOX [76]. Discher and co-workers used cryogenic electron microscopy to visualize the gradual breakdown of a polymersome from a spherical vesicle of approximately 200 nm diameter, to the gradual development of pores in the polymer membrane to eventual complete unraveling of the particle into worm-like micelles and spheres [76]. More recently, Chen et al. prepared pH-sensitive polymersomes for the stable encapsulation of Pac and DOX in an acid-labile polycarbonate polymer [77]. Virtually no release of either drug was observed at pH 7.4, however, acidic conditions (pH 4.0) yielded greater than 50% release of both agents within 12 h. Release rates were slightly faster for DOX over Pac, higher than what was observed for the singly formulated DOX particle, suggesting that Pac release was leading to vesicular membrane disruption and augmented DOX release. It was postulated that Pac release may have also been slowed by polymer micelle formation during degradation that created an additional reservoir for Pac. future science group

Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy More recently, transtuzumab (Herceptin)labeled nanocapsules containing DOX and Pac were reported [78]. Poly(vinyl alcohol) (PVA) and super paramagnetic iron oxide particles were emulsified in the presence of the active agents. To the emulsion was added a thiol-functionalized poly(methacrylic acid) polymer to render the PVA shell sensitive to pH changes and provide sites for transtuzumab conjugation. Drug release profiles of both agents were sensitive in varying degrees to the amount of poly(methacrylic acid) entangled in the PVA shell, with Pac showing greater sensitivity to pH-dependent polymer degradation due to its localization within shell of the nanocapsule. The observed release behaviors of either agent were not significantly different from that observed for singly formulated particles, suggesting that neither agent interferes with the release of the other. CombiPlex® approach to anticancer drug combinations Despite the wide variety of carrier matrices, drug combinations and formulation techniques to produce novel multidrug carriers for cancer treatment, few of the examples above account for all the criteria that determine whether a dualdrug particulate carrier will have optimal efficacy in vivo. We have discussed the importance of adequate drug loading and tunable release properties to ensure the drug carrier arrives at the site of action with sufficient bioavailable payload to exert maximal effect on the tumor, however, few studies reported above have carefully considered the potential for drugs to act synergistically and more importantly, how to achieve that activity in vivo. Without due attention to drug interactions, conditions may be created in which drug antagonism can occur. An example of such a result was provided by a liposomal formulation of DOX and vincristine [79]. While administration of free DOX at 10 mg/kg to MDA435/LCC5 tumor-bearing mice led to a tumor growth delay of 16 days, co-encapsulated DOX/vincristine (dosed at 10/2.5 mg/kg) was less effective with a tumor growth delay of 12.5 days, despite the addition of vincristine to the formulation. Analysis of this result found that while the initial formulation encapsulated the two agents at 4:1 ratio of DOX:vincristine, vincristine was less stable within the formulation and would rapidly leak from the liposome to less than 33% of its original concentration over 24 h, leading to a change in the drug:drug ratio from 4:1 to nearly 20:1. Subsequent cytotoxicity studies confirmed while the 4:1 ratio exhibited nearly 1000-times future science group

| Review

greater cytotoxicity of DOX over DOX alone, the 20:1 ratio was in fact less efficacious than the single agent. The reduced in vivo efficacy, therefore, appeared to be a result of generation of antagonistic drug ratios within the liposome. Considerable evidence points to the importance of molar drug ratios in determining whether a combination will act synergistically, additively or antagonistically in vivo [80]. The development of multidrug carriers should therefore consider both the amount and ratio of the drug combination to maximize synergy and avoid antagonism, while ensuring that the carrier system is capable of maintaining the identified ratio in vivo. The CombiPlex® approach is a system of dualdrug screening, formulation and in vivo evaluation that produces multidrug particulate systems with synergistic activity in vivo. The goal of drug screening is to identify drug:drug ratios that exhibit synergistic cytotoxicity against a wide variety of cell lines at high cell kill levels. Figure 3 schematically outlines the basic procedures of the screening method: n Use of robotics and combinatorial methods to generate numerous cytotoxicity experiments analyzing the effect of drug ratio, dose and cancer cell type on the observed cytotoxicity; Mathematical ana­lysis of the dose response matrix through the median effect method [81] to identify ratios and dose levels with synergistic, additive and antagonistic cytotoxicity;

n

Generation of a ‘heat map’ at high fraction affected levels that graphically depicts ratios and cancer cell types exhibiting strong synergistic activity.

n

Drug synergy interactions have been analyzed by this method for several drugs currently used in combination chemotherapy. We have found that widely used combinations such as cisplatin and irinotecan [82], irinotecan and floxuridine [83,84], gemcitabine and cisplatin [85], daunorubicin and cytarabine [86,87] and several others all demonstrate drug ratio dependence for synergistic, additive and antagonistic antineoplastic activity [86,88]. Using the in vitro drug combination screening informatics as a guide to select and test suitable drug:drug ratios for the cancer type of interest, iterative formulation activities produce drug carriers that coordinates release of both agents such that the drug:drug ratio is maintained in vivo. Such optimization may require meticulous and www.future-science.com

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Review | Liboiron & Mayer Custom automated screening

Cell-based assays

Dose– response matrix

Data analysis options

Surface response

Median effect

Isobologram

Median effect matrix

Identified synergistic range

Synergistic ratio identification

Antagonistic Additive Synergistic

Figure 3. Schematic diagram of the CombiPlex® drug synergy screening method. Reproduced with permission from [85] .

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Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy iterative formulation of the drug carrier and in vivo pharmaco­k inetic experimentation. For example, Tardi et al. tuned the in vivo drug release profile of irinotecan to that of floxuridine by exploiting the former drug’s drug release sensitivity to the proportion of Chol in the liposomal membrane [89]. Formulation optimization also can be assisted through thorough physicochemical characterization of carrier structure, and the drug–excipient and drug–drug interactions that may have an effect on the retention of drugs within the particle [90–92]. Through careful drug screening and formulation optimization, substantial improvements

in efficacy can be observed against various types of cancer, as shown in Figure 4 for CPX351. Packaging of cytarabine and daunorubicin at the previously identified synergistic molar ratio of 5:1 led to dramatic improvement in survival of WEHI-3B and CCRF–CEM inoculated mice over the free drug cocktail both at maximum-tolerated and ratio-matched doses (F igure  4A  & B , respectively) [87]. The importance of the drug ratio is illustrated in Figure  4C in which the overall survival of P388 ascites tumor-bearing mice is greater for those receiving the formulation at a 5:1 ratio, rather than 3:1 despite the lower ratio actually

80

80 Survival (%)

100

Survival (%)

100

60 40 20 0

| Review

60 40 20

0

15

30 45 60 75 Days post cell inoculation

0

90

20

40 60 Days post cell inoculation

80

Survival at day 55 (%)

100 80 60 40 20 0 Cytarabine

Saline

1:1

3:1

5:1

12:1

N/A 5.4 mg/kg 10 mg/kg 10 mg/kg 15 mg/kg

Daunorubicin N/A 12.5 mg/kg 7.7 mg/kg 4 mg/kg

3 mg/kg

Figure 4. In vivo efficacy improvements achieved through maintenance of a synergistic 5:1 ratio of cytarabine and daunorubicin within CPX-351. (A) Survival of WEHI-3B ascites tumor-bearing CD-1 nude mice (n = 6 per group) after intravenous treatment with saline (blue), maximum tolerated doses (MTD) of dose-pushed (300:4.5 mg/kg, purple) or ratio-matched (12:5.3 mg/kg, green) cytarabine:daunorubicin or CPX-351 (12:5.3 mg/kg, red). (B) Survival of CCRF–CEM tumor-bearing SCID/Rag2M mice (n = 6 per group) after treatment with saline (purple), MTD of dose-pushed (300:4.5 mg/kg, green) or CPX-351 at 6.3:2.5 mg/kg (red) or MTD (10:4.4 mg/kg, blue). (C) Survival of P388 ascites tumor-bearing BDF-1 mice (n = 6 per group) after treatment with saline or co-encapsulated cytarabine:daunorubicin liposomes. Formulations were dosed at MTD with the exception of 5:1, dosed at 0.8 MTD. Reproduced with permission from [87] .

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Review | Liboiron & Mayer administering a higher dose of daunorubicin (4 vs 7.7 mg/kg, with equivalent doses of cytarabine for ratios 5:1 and 3:1, respectively). CPX351 has also demonstrated enhanced selective in vitro cytotoxicity for acute myeloid leukemia (AML) rather than normal cell progenitors [93], through selective uptake of the intact liposome in bone marrow xenografts [94], neither of which are observed through conventional ‘cocktail’ administration of the two active agents without the drug carrier. Table 5 lists formulations developed through the CombiPlex platform, two of which represent, to our knowledge, the first dual-drug particulate-carrier systems to proceed to clinical trials. A wide variety of antineoplastic agents have been successfully formulated with particulate carriers at identified synergistic ratios. It is worth noting that the CombiPlex platform has also been extended to hydrophobic agents such as Pac and docetaxel through synthesis of prodrug nanoparticles [95]. Attachment of nonpolar anchor molecules to the drug of interest changes the release properties from drugdependent to anchor-dependent, such that through varying the degree of hydrophobicity of the anchor, release rates of highly chemically disparate drugs such as Pac and gemcitabine can be normalized and the synergistic ratio maintained in vivo. Tangible improvements in both safety and efficacy of CombiPlex formulations over conventional cocktail therapies have been demonstrated in several clinical trials [96–99]. CPX351, a CombiPlex formulation of cytarabine and daunorubicin for treatment of AML, was recently advanced into Phase III clinical testing. Two separate dual arm Phase II trials demonstrated significant overall survival improvement in newly diagnosed secondary AML and high risk first relapse patients over conventional 7+3 cytarabine:daunorubicin induction treatment and various salvage therapies, respectively [97,98]. It is hoped that the

careful design and optimization of this platform will lead to improvements in the current treatment of AML and a definitive validation of the value of ratiometric, synergistic delivery of antineoplastic agents to tumor sites. Future perspective Significant technical advancements have been made in the application of nanoscale delivery systems to drug combinations. Technology platforms now exist with which drugs exhibiting disparate physicochemical properties can be coformulated and their biodistribution properties coordinated so as to optimize the efficacy of the combination, whether this be via sequential or simultaneous exposure to the target disease site. The vast amount of work done to date in this context has been with anticancer drug combinations, and more specifically, with combinations of cytotoxic agents typically utilized in conventional combination chemotherapy regimens. More recently, attention has been given to combinations where a conventional cytotoxic drug is combined with a biological modulating agent or molecularly targeted therapeutic. As we look forward, it is clear that increased efforts should be focused on delivering combinations of molecularly targeted agents [100,101]. The redundant and/or compensatory feedback pathways associated with most types of cancer will most certainly require well-coordinated inhibition of multiple signaling pathways in tumor cells in order to achieve optimal efficacy. The applicability of existing drug-carrier technologies to the various chemical classes of such agents remains to be determined and will no doubt pose additional formulation challenges. However, the ability of delivery systems to make a meaningful impact in the development of anticancer drug combinations will depend on their successful application to combinations of molecularly targeted agents, given the prevalent interest in such therapeutics compared with traditional cytotoxics.

Table 5. Dual-drug formulations developed via the CombiPlex® platform for in vivo maintenance of synergistic drug:drug ratios. Formulation

Active agents

Ratio

Target indication

Status

CPX-1

Irinotecan Floxuridine Cytarabine Daunorubicin Irinotecan Cisplatin

1:1

Colorectal cancer

Phase II

5:1

Acute myeloid leukemia

Phase III

7:1

Non-small-cell lung cancer

Preclinical

CPX-351 CPX-571

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Ther. Deliv. (2014) 5(2)

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Nanoscale particulate systems for multidrug delivery: towards improved combination chemotherapy A significant number of previous drug-combination-delivery formulations utilized cytotoxic chemotherapy agents in combination with modulators of individual proteins associated with drug resistance (e.g., P-gp and Bcl-2). However, efforts to increase chemosensitivity using this approach have not yielded promising clinical results to date regardless of whether either of the agents were formulated in a delivery vehicle or not. Consequently, it may be that the development of effective chemosensitizer regimens will require the simultaneous use of multiple such modulators. For example, one may require the simultaneous inhibition of drug efflux pumps, prosurvival proteins and cell cycle checkpoints to effectively increase the chemosensitivity of tumor cells [102]. Finally, a future challenge facing the delivery of drug combinations is the ability to increase the specificity and efficiency of drug delivery to the disease site. Passive tumor and infection site delivery of nanoscale drug carriers has been widely relied on for shifting the drug distribution profile in favor of the disease site, however the therapeutic improvements achieved using this strategy, while meaningful and important, have been generally incremental. Whether the addition of specific targeting moieties onto the surface of delivery vehicles can introduce a new level of disease site selectivity and drug delivery efficiency remains to be seen. However, there are promising signs that nanomedicines incorporating such features are pharmaceutically feasible and may increase therapeutic activity.

| Review

In summary, the prevalent and expanding use of drug combinations, particularly for cancer treatment represents a significant opportunity for drug-delivery applications. The need to control how individual agents of a combination treatment regimen are exposed to disease cells both in terms of concentrations and time appears well matched with the pharmacological benefits provided by nanoscale drug-delivery vehicles. In addition, co-formulated nanomedicines may be particularly useful in expediting the clinical evaluation of drug combinations by avoiding the redundant loop of testing agents first individually before evaluating their activity as a combination. This may be especially important in cases where one or more of the agents has minimal therapeutic activity by itself. Regardless, there will clearly be an abundant and ever-renewing source of drug combinations to select from for drug-delivery applications. Financial & competing interest disclosure The authors are paid employees of Celator Pharmaceuticals Corp based in Vancouver BC, Canada, with corporate headquarters at Celator Pharmaceuticals Inc. in Ewing, NJ, USA. The authors disclose ownership and stock option interests in the company. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Background Multidrug particulate carriers can be classified under three broad groups based on whether the goal is efficacy improvement via sensitization, sequential delivery or simultaneous drug exposure. Combinations of chemotherapeutics & drug sensitizers „„

„„

Delivery of direct or nucleotide chemotherapy sensitizers require substantially different formulation methods and particle compositions.

Formulation of nucleotide-based sensitizers with antineoplastic agents is complicated by the narrow formulation search space imposed by the nucleotide agents. Sequential delivery of multiple agents „„

„„

True sequential release of multiple agents in vivo remains difficult; few examples exist that demonstrate rational design of a one–two exposure of two agents from a single particle.

Reported delivery systems limited to antineogenesis agents with a second antineoplastic agent. Limited understanding of drug sequencing at the cellular level hinders development. Simultaneous delivery of multiple agents „„

„„

Harnessing drug–drug synergy in vivo requires coordination of drug release rates at the target site.

„„

Drug interactions in vivo remain a secondary consideration in the development of dual-drug particulate carriers.

„„

The only dual-drug-carrier systems that have proceeded to the clinic were developed with the implications of drug–drug interactions, both positive and negative, clearly in mind.

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Review | Liboiron & Mayer References Papers of special note have been highlighted as: n of interest nn of considerable interest 1

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