A Polymeric Nanoparticle Encapsulated Small-Molecule Inhibitor of Hedgehog Signaling (NanoHHI) Bypasses Secondary Mutational Resistance to Smoothened Antagonists

The Hedgehog (Hh) signaling pathway plays a critical role in development as well as in mature tissue homeostasis (1). Aberrant activation of the Hh pathway is commonly observed in many human cancers, and it is implicated in tumor initiation as well as tumor progression (2, 3). Hh activation in human cancers can occur either via a ligand-independent mechanism, as is observed in so-called Gorlin syndrome cancers (basal cell carcinoma and medulloblastoma), or by a ligand-dependent mechanism, which has been implicated in many solid tumors (4, 5). Ligand-independent pathway activation is usually a consequence of loss-of-function mutations in the gene encoding the Hh inhibitory receptor Patched (Ptch), or less commonly, a gain-of-function mutation in the gene encoding the essential Hh signal transduction receptor Smoothened (Smo; refs. 6–8). In contrast, mutational activation of the pathway is rare in endodermal tumors such as pancreatic cancer that produce aberrantly high levels of Hh ligand (2). As recent evidence has shown, most of the pathway activity in such cancers seems to be restricted to the juxtatumoral stromal cells rather than the neoplastic cells per se (9, 10). Irrespective of the mechanism of pathway activation, derepression of Smo from Ptch initiates an intracellular cascade that culminates in the nuclear translocation of Gli transcription factors, and the major transcriptional activator in human cancers seems to be Gli1, a product of the GLI1 oncogene (11).

In the area of clinical oncology, small-molecule antagonists of Hh signaling have emerged as a more promising targeted approach to cancer therapy. Recently, there has been compelling evidence to suggest that cancer cells can acquire resistance to Smo antagonists through secondary mutations in Smo. Most notably, Yauch and colleagues recently reported on a patient with metastatic medulloblastoma, who initially had a dramatic response to GDC-0449, but subsequently relapsed with refractory disease (12). Sequencing of the relapsed tumor DNA identified a secondary mutation in an extracellular loop of the Smo heptahelical bundle (Smo^D473H), which abrogates the binding of GDC-0449. Strikingly, in a Ptch^−/+; p53^−/− mouse model of medulloblastoma selected in vivo for resistance to GDC-0449, an acquired Smo^D477G alteration was identified, which is orthologous to the aspartic acid residue at position 473 in humans and similarly disrupts antagonist binding. Of note, neither mutation has an impact on the overall level of Hh activation, suggesting that these are not independently oncogenic. Independent experiments in murine medulloblastoma models have also reported comparable acquired mutations in Smo that confer Hh inhibitor resistance (13, 14). In addition, other mechanisms of resistance to Smo antagonists have also been reported, including amplification of Gli oncogenes that occur downstream of the Smo receptor (13, 14), thus allowing cancer cells to bypass Hh blockade by the current compendium of Smo antagonists.

In light of this emerging evidence on mechanisms of secondary resistance to Smo antagonists, there is a pressing need to identify a new generation of Hh inhibitors that block signaling downstream of Smo. In 2009, Hyman and colleagues identified a series of 4 Hh pathway inhibitors (HPI 1–4), which block signaling at diverse points downstream of Smo, including Gli processing, stability, and trafficking to the primary cilium (15). One of these compounds, HPI-1, is a potent antagonist of both endogenous activator Gli proteins (Gli1/2) and can also abrogate Hh signaling in the setting of exogenous Gli overexpression. On the basis of its mechanism of action, we can postulate that HPI-1 will circumvent acquired mutational resistance to conventional Smo inhibitors.

Despite the promising in vitro findings, however, the in vivo translation of HPI-1 is likely to be hampered by its highly lipophilic nature and poor aqueous solubility, thereby impairing systemic bioavailability. To harness the full therapeutic potential of HPI-1, we have generated a polymer nanoparticle-encapsulated formulation of HPI-1 (NanoHHI), which overcomes the barriers to systemic bioavailability. NanoHHI has been engineered using [poly(lactic-co-glycolic acid); (PLGA)] conjugated with polyethylene glycol (PEG), both of which are considered as generally regarded as safe components by the U.S. Food and Drug Administration (16). NanoHHI shows strikingly higher systemic bioavailability than the HPI-1 alone upon parenteral administration, with no apparent histopathologic or biochemical evidence of toxicities in mice. Of importance, NanoHHI blocks Hh signaling in cells with ectopic expression of the human Smo^D473H allele and significantly inhibits the in vivo growth of murine medullobastoma allografts harboring the acquired murine Smo^D477G mutation, both of which confer resistance to targeted Smo antagonists (12). NanoHHI also inhibits the growth of orthotopic human pancreatic cancer xenografts that harbor a wild-type Smo allele, by potentiating the effects of gemcitabine in the orthotopic milieu. Thus, NanoHHI represents a promising new therapeutic formulation for treatment of human cancers with primary or secondary resistance to Smo antagonists.

PLGA conjugated with PEG, that is, PLGA-PEG (5050 DLG, mPEG 5000) was purchased from Lakeshore Biomaterials. Dichloromethane, acetone, and polyvinyl alcohol PVA_18k (87%–89% hydrolyzed) were obtained from Sigma Aldrich. HPI-1 was synthesized according to the reported procedure (15); the nanoparticulated formulation NanoHHI was stored as a lyophilized powder at 4°C and dissolved in PBS on the day of use. Gemcitabine (NetQem LLC) was stored at 4°C and dissolved in sterile NaCl (0.9% w/v) on the day of use. HhAntag (Genentech), a parental drug of the lead clinical compound GDC-0449 (17), was freshly formulated as a suspension in 0.5% methylcellulose/0.2% Tween-80 (MCT).

Pa03C (LZ10.7), a low-passage metastatic human pancreatic cancer cell line, was cultured as described (18); the authentication of this cell line was based on representative validation of previously described whole exome mutational profiling data (19). Either wild-type Smo or Smo^D473H mutant cDNA was cloned into pRK5 mammalian expression vector, and stable clones of HEK293 cells expressing empty pRK5, pRK5-Smo^wt, or pRK5-Smo^D473H were generated for Hh reporter assays. Murine medulloblastoma allografts derived from Ptch^−/+; p53^−/− mice harboring either Smo^wt or Smo^D477G alleles were generated as described previously for in vivo studies comparing HhAntag and NanoHHI (12).

NanoHHI was prepared with a modification of the oil-in-water (o/w) emulsion solvent evaporation method (20). Briefly, 3 g of PLGA-PEG and 60 mg of HPI-1 was dissolved in 30 mL of dichloromethane and acetone (8:2), and the resulting solution was added to 0.4% polyvinyl alcohol (150 mL). The mixture was sonicated for 3 minutes with stirring (20W, 4°C). The suspension was stirred at room temperature for 4 hours to evaporate organic solvents, and complete evaporation of organic solvents was achieved by using the rotary evaporator. The resulting suspension was ultracentrifuged at 40,000 rpm for 45 minutes. The precipitated nanoparticle pellet was washed 3 times with ultrapure water and resuspended in ultrapure water by mild sonication and centrifuged at 3,000 rpm for 5 minutes to remove large aggregates or any nonencapsulated drug. The supernatant NanoHHI was flash frozen on dry ice and lyophilized to obtain a dry NanoHHI powder amenable to long-term storage. Drug loading and loading efficiency were determined by dissolving dried NanoHHI in 80% methanol in water and quantifying by high-performance liquid chromatography (HPLC). Drug loading percentage (L) was calculated as follows: L = (m_drug/m_total) × 100, where m_drug and m_total are the mass of drug and loaded nanoparticles (NanoHHI), respectively. Loading efficiency (E) was calculated as follows: E = (m_encap/m_drug) × 100, where m_encap and m_drug are the mass of drug encapsulated and the total mass of drug initially loaded, respectively.

Size analysis of NanoHHI was done with a Malvern Zetasizer (Malvern), and transmission electron microscopy images obtained with a Hitachi 7600 (Hitachi Ltd.). Release kinetics studies were done with the dialysis method, as described (21). Briefly, NanoHHI containing 10 mg equivalent of HPI-1 dispersed in 5mL ultrapure water was transferred into a dialysis bag with a molecular cutoff value of 12 kDa. The bag was suspended in 100 mL of release medium [50% (v/v) of ethanol, water] in a container, with stirring at 50 rpm at 37°C. A 300 μL aliquot was withdrawn at predetermined time intervals and replaced with fresh release medium. Finally these samples were analyzed by HPLC with a Waters HPLC system. As HPI-1 is poorly soluble in water, ethanol was used in the release medium to ensure sink conditions.

To compare the in vivo pharmacokinetics of NanoHHI versus free HPI-1, we conducted 3 independent studies, each via a different route of administration: First, for the i.p. route, 2 cohorts of 3 mice each were administered a single i.p. injection of either 25 mg/kg free HPI-1 suspended in corn oil or 25 mg/kg NanoHHI equivalent. Second, for the i.v. route, 2 cohorts of 4 mice each were administered either 30 mg/kg free HPI-1 in 40% ethanol or 30 mg/kg NanoHHI equivalent. Third, for the oral bioavailability study, 2 arms of 4 mice each were administered a single per oral dose of either 30 mg/kg free HPI-1 suspended in corn oil or 30 mg/kg NanoHHI, via oral gavage. Finally, to assess the ability of NanoHHI to cross the blood–brain barrier (BBB), 2 cohorts of 3 mice each were administered a single intravenous injection of 30 mg/kg NanoHHI (this study was done in replicate to collect terminal brain samples at 2 different time points following injection). All of the earlier studies were conducted using nontumor bearing CD1 mice. Blood samples (50 μL) were obtained from the mice at predetermined time intervals postinjection, with EDTA-coated Microvette CB300 capillary tubes (Braintree Scientific). The plasma was separated by centrifuging the blood samples at a speed of 1000 × g for 5 minutes. Brain samples were collected immediately following euthanasia at 10 and 30 minutes postinjection.

HPI-1 levels were estimated with liquid chromatography/tandem mass spectrometry (LC/MS-MS). Tissue homogenates were prepared at a concentration of 200 mg/mL in plasma before extraction. HPI-1 was extracted by acetonitrile:n-butyl chloride (1:4, v/v) and separated on a Waters X-Terra MS C₁₈ (50 × 2.1 mm, 3.5 μm) column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/minute for 5 minutes. Plasma calibration curves were prepared over the range of 0.01 to 2 μg/mL or 0.06 to 12 μg/g, with a 1:1,000 dilution being accurately quantitated. Concentration–time data were evaluated using a noncompartmental approach with individual profiles (WinNonlin Professional, version 5.2 software; Pharsight Corporation).

All small animal experiments described conformed to the guidelines of the Animal Care and Use Committee of Johns Hopkins University. Mice were maintained in accordance with the guidelines of the American Association of Laboratory Animal Care.

Flanks of 5 to 6 weeks old male athymic nu/nu mice (Harlan Laboratories) were injected with a single-cell suspension (2 × 10⁶ cells) of either Smo^WT; Ptch^+/−; Trp53^−/− or Smo^D477G; Ptch^+/−; Trp53^−/− in a total volume of 200 μL PBS/Matrigel [BD Biosciences; 1:1 (v/v), prechilled to 4°C]. One week after the injection of tumor cells, subcutaneous tumor volumes (V) were measured with digital calipers (Fisher Scientific) and calculated using the formula as follows: V = 1/2(ab²), where a is the biggest and b is the smallest orthogonal tumor diameter. Mice with successfully engrafted. Smo^WT or Smo^D477G allografts were then randomized into 3 cohorts of 6 animals each and administered one the following regimens: (i) void PLGA-PEG nanoparticles, (ii) NanoHHI at a dose of 30 mg/kg i.p. twice daily, and (iii) HhAntag at a dose of 100 mg/kg orally, once daily. NanoHHI dosing of 30 mg/kg was chosen after multiple dose tolerance study (data not shown). The allografted mice were monitored daily for any signs of toxicity and behavioral abnormalities during the treatment. Tumor size and body weight were measured every other day. At the end of treatment, allografts were harvested and preserved for mRNA extraction and quantitative real-time PCR analysis of Hh gene targets.

The generation of orthotopic Pa03C human pancreatic cancer xenografts by surgical implantation in athymic mice has been described previously by our group (18). Three weeks after surgical orthotopic implantation, the presence of intrapancreatic primary tumors was confirmed by ultrasound scan (Vevo660; VisualSonics). Twenty-eight mice with demonstrable primary xenografts were then randomized into 4 cohorts, with 7 mice per arm, as follows: (i) void PLGA-PEG nanoparticles, (ii) NanoHHI at a dose of 30 mg/kg i.p. twice daily, (iii) gemcitabine at a dose of 20 mg/kg i.p. twice weekly, or (iv) the combination of NanoHHI (30 mg/kg i.p. twice daily) and gemcitabine (20 mg/kg i.p. twice weekly). Both agents were administered for a period of 4 weeks. At the end of therapy, primary tumors were carefully excised from the pancreas and weighed. Portions of treated xenografts were also preserved for mRNA extraction and qRT-PCR for Hh gene targets (primer sequences for human and mouse genes are readily available upon request).

Transmission electron microscopy (Fig. 1A) and dynamic light scattering studies (data not shown) confirmed that NanoHHI has an average diameter of 60 nm. In contrast to the extremely poor aqueous solubility of free HPI-1, NanoHHI was well dispersed to form a uniform suspension in aqueous media (Fig. 1B). HPI-1 was loaded efficiently in the void PLGA-PEG nanoparticles [2% (w/w) loading; 90% encapsulation efficiency]. The in vitro release kinetics of HPI-1 from NanoHHI was assessed over an approximately 4-week period by dialysis method, using 50% v/v ethanol as release medium to ensure sink conditions due to the nearly complete insolubility of free HPI-1 in aqueous media (Fig. 1C). A small burst release in the initial 24 hours was observed (cumulative ∼30%), followed by a relatively slow release over 3 days (cumulative ∼70%), with complete drug being released by day 25.

We compared the bioavailability of NanoHHI to free HPI-1 dissolved in corn oil, following administration of a single equivalent dose of either formulation through the i.p. route in nontumor-bearing mice (Fig. 1D). Relevant pharmacokinetic parameters, including C_max, T_max, and area under curve (AUC) are tabulated in Supplementary Table S1. There was an appreciable difference in the bioavailability of intraperitoneal HPI-1 between the free drug and nano formulation, with NanoHHI showing an AUC_0–∞ value twice that of free HPI-1 in corn oil (52 ± 45 μg h/mL vs. 26 ± 12 μg h/mL). We also attempted to compare the pharmacokinetics of i.v. NanoHHI versus free HPI-1 dissolved in 40% ethanol as an excipient; however, the mice receiving intravenous ethanolic HPI-1 succumbed within 1 to 2 minutes of injection, likely from the excipient-related toxicity. The AUC_0–∞ value of intravenous NanoHHI alone was 268 ± 98 μg h/mL (Supplementary Fig. S1A and Table S1). A more pronounced improvement in bioavailability was observed after oral administration of 30 mg/kg HPI-1 equivalents, with the NanoHHI AUC_0–∞ value being 4-times higher than that of free HPI-1 in corn oil (17 ± 31 μg h/mL vs. 4.2 ± 1.4 μg h/mL; Supplementary Fig. S1B and Table S1). Thus, we were able to establish the feasibility of administering NanoHHI through multiple routes, with a 2- to 4-fold improved AUC values for the 2 modes (intraperitoneal and oral) where an equimolar comparison could be made versus free HPI-1.

A potential clinical application of Gli-targeted Hh agents will be in the setting of medulloblastomas that have acquired secondary mutations in SMO; thus, establishing tractable delivery of systemic NanoHHI past the BBB is paramount in that respect. Following single-dose intravenous administration of NanoHHI, HPI-1 was readily detectable in brain tissue at 3.9 ± 2.1 μg/g after 10 minutes and 1.4 ± 0.4 μg/g at 30 minutes after injection (Supplementary Fig. S1C).

In vitro Hh reporter assays were conducted using HEK-293 cells expressing either wild-type pRK5-Smo^WT or the Smo antagonist–resistant binding site mutant pRK5-Smo^D473H construct (Fig. 2A). Whereas both HPI-1 and cyclopamine were able to significantly inhibit reporter activity in the setting of wild-type Smo, only HPI-1 resulted in significant downregulation of reporter activity in HEK-293 cells expressing the mutant Smo^D473H. These results confirmed the ability of our indigenously synthesized free HPI-1 to bypass the Smo binding site mutation in vitro.

We then proceeded to in vivo assays using the NanoHHI formulation, comparing it to the potent and selective Smo antagonist, HhAntag. In subcutaneous medulloblastoma allografts derived from Smo^WT; Ptch^+/−; Trp53^−/− tumors, both NanoHHI and HhAntag inhibited in vivo growth to a comparable degree (Fig. 2B, left). In contrast, in allografts derived from Smo^D477G; Ptch^+/−; Trp53^−/− tumors, there was a marked difference in efficacy between NanoHHI and HhAntag (Fig. 2B, right). Although even HhAntag had a significant negative impact on allograft growth (P < 0.01), NanoHHI essentially flattened the tumor growth curve (P < 0.001). The partial response seen with HhAntag in the setting of a binding site mutation may reflect residual binding capability to the mutant Smo receptor, and/or off-target effects, an effect not observed with the clinical grade compound (14). In treated allografts, qRT-PCR for MmGli1 transcripts mirrored the efficacy data (Fig. 2C), with significant MmGli1 downregulation observed in both treatment arms for Smo^WT; Ptch^+/−; Trp53^−/− tumors (P < 0.001) but only in the NanoHHI arm for Smo^D477G; Ptch^+/−; Trp53^−/− tumors (P < 0.001; all of the transcript results were normalized to levels observed in allografts treated with void polymer). Because HPI-1 is a direct inhibitor of Gli function and acts downstream of Smo, we evaluated the impact of NanoHHI or HhAntag treatment in a panel of recently described Gli target genes in murine medulloblastoma including Akna, Cltb, and Olig2 (Fig. 2D; ref. 22). For these analyses, only the Smo^D477G; Ptch^+/−; Trp53^−/− allografts were assessed, and confirmed the ability of NanoHHI, but not the targeted Smo inhibitor, to significantly block bona fide Gli target gene expression in the treated tumors. In this study, we observed no significant alterations of body weight in any of the NanoHHI treatment arms, whereas the HhAntag-treated mice showed approximately 10% body weight loss at the end of treatment (Supplementary Fig. S1D).

Gorlin syndrome tumors with cell autonomous Hh signaling comprise only a minor fraction of cases in the universe of solid cancers, with ligand-dependent Hh signaling being the most common mechanism of activation by far. In this latter instance, much of the signaling is paracrine in nature, and the nonneoplastic stromal cells may be significantly less likely to acquire secondary mutational hits during therapy. Nonetheless, given the demonstrable efficacy of Hh blockade in preclinical models of ligand-dependent cancers, we explored the in vivo effects of NanoHHI in an orthotopic human pancreatic cancer xenograft model. In Pa03C xenografts, we found that NanoHHI monotherapy had no significant effect on orthotopic tumor growth (P = 0.58), mirroring what has been reported with cyclopamine and other Smo antagonists (18, 23). In contrast, NanoHHI in combination with gemcitabine significantly improved tumor growth inhibition over gemcitabine alone (P = 0.018; Fig. 3A). We have previously shown that cyclopamine and other Smo antagonists deplete the primary tumor of cells expressing aldehyde dehydrogenase (ALDH), a credentialed marker of tumor-initiating cells (i.e., cancer stem cells; refs. 24, 25), even when there is no impact on gross tumor volume (18, 23). We confirmed that NanoHHI, either as a single agent, or in combination with gemcitabine, can cause a marked decrease in ALDH-expressing cells within the orthotopic Pa03C xenograft (Fig. 3B). Furthermore, we also confirmed that stroma-derived MmGli1 was significantly reduced in both NanoHHI single-agent (P < 0.05) and combination therapy arms (P < 0.001; Fig. 3C), consistent with inhibition of Hh signaling in the murine stromal compartment. In contrast, gemcitabine alone had no significant impact on MmGli1 levels. We also examined the levels of HsCMYC, a key transcriptional regulator in pancreatic cancer, and found no significant effects of NanoHHI compared with void nanoparticles (notably, gemcitabine resulted in significant downregulation of expression in both single agent and combination therapy arms; Supplementary Fig. S2A). The expression of HsHES1, a Notch pathway target gene, however, was significantly downregulated by NanoHHI, consistent with the recent data on noncanonical regulation of HES1 expression by Hh signaling (26). Finally, we examined the levels of MmNestin, a marker of neoendothelial cells in the murine stroma (27) and found significant downregulation in both single-agent and combination therapy arms, consistent with the known antiangiogenic effects of Hh blockade in tumors (Supplementary Fig. S2B).

The preclinical and emerging clinical use of the small-molecule Hh blockade has shown the surprising resilience of nonneoplastic Hh-dependent compartments (e.g., bone marrow) to this class of agents (28). Thus, there seems to be a therapeutic window, wherein depriving Hh signals to cancer cells themselves, or to the microenvironment, can inhibit tumor growth without major systemic deleterious effects. To determine whether a similar safety profile might exist for NanoHHI, particularly as the active pharmaceutical ingredient (API) has a distinct target (Gli) from the currently studied Smo antagonists, we did a panel of laboratory assays on NanoHHI-administered mice. In both the medulloblastoma allograft and the orthotopic pancreatic cancer xenograft experiments, which lasted for a period of 2 to 4 weeks, we observed no loss of body weight in any of the NanoHHI treatment arms (see Supplementary Fig. S1D and Fig. 3D). We also examined the histopathology of major viscera in NanoHHI-treated mice, which did not reveal any microscopic abnormalities (data not shown). Finally, we examined a panel of laboratory parameters, including hematologic (red blood cell, white blood cell, platelet counts, and hemoglobin), renal and liver function tests in NanoHHI mice compared with vehicle-treated animals (Supplementary Fig. S3) and observed no significant differences in any of the results.

Aberrant activation of the Hh pathway is observed in many solid and hematologic cancers, generating considerable excitement about targeted inhibition of this pathway. As recent data have shown, patients whose tumors harbor mutational loss of Patched can experience dramatic responses to a class of orally bioavailable Smo antagonists, of which GDC-0449 is farthest along in clinical development (29, 30). Nonetheless, several mechanisms of secondary resistance to Hh blockade in tumors have emerged recently, most notably the acquisition of somatic mutations of Smo, which render the receptor incapable of binding to GDC-0449 and other antagonists of this class (12, 13). The acquisition of secondary resistance to targeted therapies is certainly not unprecedented, with chronic myelogenous leukemia being one of the better known examples where this phenomenon occurs in response to imatinib (31).

There is an urgent impetus for finding alternative targets in the Hh pathway, particularly those that function downstream of Smo. The Gli transcription factors represent ideal targets for this purpose, in light of their role as the final intracellular mediator of Hh signal transduction, both ligand dependent and independent. Given that Gli1 can be either primarily amplified (e.g., in gliomas, from which this gene inherits its eponymous designation; ref. 32) or secondarily amplified/overexpressed in the setting of Hh inhibitor therapy (13), a pharmacologic strategy targeting Gli could have tangible benefits. Recent small molecule screens have indeed identified such lead candidates, of which a panel of 4 HPIs has been extensively characterized in vitro vis-à-vis their mechanisms of action (15). Of these, HPI-1 is a potent inhibitor of Gli function, including exogenously expressed Gli1 in cells, underscoring its potential as an agent of choice in cancers that acquire resistance to Smo antagonists. A major pitfall of the parental HPI-1 compound is its lipophilicity, which makes systemic delivery and adequate bioavailability a challenge.

In this article, we present the initial characterization of a novel nanoparticle formulation of HPI-1 using the polymer PLGA as a backbone. PLGA has been extensively used as a vehicle for enabling systemic delivery of therapeutic agents that otherwise require noxious excipients like cremophor or ethanol and has been conferred a generally regarded as safe status by the U.S. Food and Drug Administration (16, 33). Although the active pharmaceutical ingredient in NanoHHI is identical to that of free HPI-1, we have showed the improved systemic bioavailability of the nanoformulation compared with the free drug upon both oral and parenteral administration in mice. In addition, we have documented the ability of intravenous NanoHHI to cross the BBB, resulting in detectable levels of the active pharmaceutical ingredient in the brain. In this study, we establish the ability of NanoHHI to bypass secondary resistance to conventional potent and selective Smo antagonists in a medulloblastoma allograft model. We have also shown that in preclinical models of pancreatic cancer, wherein the major component of Hh signaling is paracrine and acquired mutational resistance is potentially less of an issue, NanoHHI has therapeutic efficacy comparable with what has been reported for cyclopamine and related Smo antagonists (18, 23). Although we did not explicitly examine the efficacy of NanoHHI in a model of Gli overexpression (such as in a glioblastoma with GLI1 amplification), data presented in the study by Hyman and colleagues suggest that NanoHHI should show activity in this setting, based on its ability to block Gli function at multiple levels (15). Whether NanoHHI also acts in acquired Smo inhibitor resistance secondary to other mechanisms like activation of the phosphoinositide 3-kinase pathway remains to be seen.

In conclusion, we present a novel polymer nanoparticle formulation of a potent Hh inhibitor, NanoHHI, which shows in vivo ability to circumvent acquired mutational resistance to the commonly used clinical Smo antagonists. In light of its demonstrable efficacy in preclinical allograft and xenograft models of both ligand-independent and ligand-dependent Hh signaling and its ability to cross the BBB, NanoHHI has the potential to be used across a wide spectrum of tumor types. Future studies will expand the application of NanoHHI into the areas of autochthonous and orthotopic mouse models, including those generated by stereotactic injection within the central nervous system. The oral bioavailability of NanoHHI also provides an avenue for exploring a role for Hh-targeted chemoprevention in cognate systems.

No potential conflicts of interest were disclosed.

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