Recombinant human hyaluronidase PH20-mediated dermal spreading activity in mice is not altered by steroids, antihistamines, or salicylic acid.

OBJECTIVES: Drug-drug interaction studies for hyaluronidase safety assessments have evaluated only animal-derived enzyme preparations. We therefore set out to evaluate whether high-dose administration of two antihistamines, a potent corticosteroid, steroid hormone, adrenocorticotropic hormone (ACTH), or salicylic acid would alter the dispersive activity of recombinant human hyaluronidase PH20 (rHuPH20). METHODS: NCr nu/nu mice were pretreated with diphenhydramine, cetirizine, dexamethasone, estrogen, ACTH, salicylic acid, and/or neutral-buffered saline (NBS). An hour following final pretreatment, dosed mice were anesthetized with ketamine/xylazine and placed in an imaging chamber. A 120 mg/mL immunoglobulin G (IgG) solution with 0.3 µg/mL IgGDL755 (labeled IgG) was injected intradermally, with/without 2,000 U/mL rHuPH20. Fluorescent images of labeled IgG dispersion were acquired ≤20 min post injection. RESULTS: Dispersion of high-concentration labeled IgG combined with rHuPH20 was greater at all time points vs. antibody alone. At 20 min post injection (last time point), the antibody dispersion area was significantly increased with rHuPH20 vs. without rHuPH20 (p≤0.005). The relative percent increase in antibody dispersion with rHuPH20 ranged from 22.8‒106.6% over the 20-min time course, compared with the corresponding non-rHuPH20 treated groups. The area of labeled IgG dispersion was statistically similar between rHuPH20 groups pretreated with an active compound and their paired NBS pretreated controls. CONCLUSIONS: The addition of 2,000 U/mL rHuPH20 to a high-concentration antibody solution reproducibly incre-ased local antibody dispersion. Systemic pretreatment with diphenhydramine, cetirizine, dexamethasone, estrogen, ACTH, or salicylic acid did not affect the enzymatic spreading activity of rHuPH20, as measured by intradermal dispersion of labeled IgG in mice.

Parallel Accumulation of Tumor Hyaluronan, Collagen, and Other Drivers of Tumor Progression.

Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors.Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance.Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content.Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798-807. ©2018 AACR.

Recombinant human hyaluronidase PH20 does not stimulate an acute inflammatory response and inhibits lipopolysaccharide-induced neutrophil recruitment in the air pouch model of inflammation.

Hyaluronidase (Hyal) and low m.w. hyaluronan (LMW HA) fragments have been widely reported to stimulate the innate immune response. However, most hyaluronidases used were purified from animal tissues (e.g., bovine testis Hyal [BTH]), and contain endotoxin and other unrelated proteins. We tested a highly purified recombinant human Hyal (rHuPH20) and endotoxin-free HA fragments from M(r) 5,000 to 1,500,000 in the rodent air pouch model of inflammation to determine their potential for stimulation of the innate immune response. Exogenous LMW HA fragments (average M(r) 200,000) failed to induce either cytokine/chemokine production or neutrophil infiltration into the air pouch. Challenging the air pouch with LPS or BTH stimulated production of cytokines and chemokines but rHuPH20 did not, suggesting that neither PH20 nor generation of LMW HA fragments in situ stimulates cytokine and chemokine production. LPS and BTH also induced neutrophil infiltration into the air pouch, which was not observed with rHuPH20 treatment. Endotoxin-depleted BTH had much reduced proinflammatory activity, suggesting that the difference in inflammatory responses between rHuPH20 and BTH is likely due to endotoxin contaminants in BTH. When rHuPH20 was dosed with LPS, the induction of cytokines and chemokines was the same as LPS alone, but neutrophil infiltration was inhibited, likely by interrupting HA-CD44 interaction. Our results indicate that neither rHuPH20 nor its directly generated HA catabolites have inflammatory properties in the air pouch model, and rHuPH20 can instead inhibit some aspects of inflammation, such as neutrophil infiltration into the air pouch.