Hyaluronan in cytosol--microinjection-based probing of its existence and suggested functions.

Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested (∼HA120 or ∼25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.

Assessing the role of human recombinant hyaluronidase in gravity-driven subcutaneous hydration: the INFUSE-LR study.

BACKGROUND: Subcutaneous hydration has potential advantages over intravenous. Despite studies supporting the efficacy and safety of subcutaneous hydration it has not been studied extensively to date either with or without hyaluronidase. OBJECTIVES: To compare flow rate, tolerability, and safety of gravity-driven subcutaneous fluid administration with and without recombinant human hyaluronidase (rHuPH20) in healthy volunteers. DESIGN: Randomized, double-blind, placebo-controlled, within-subject trial. SETTING: Contract research organization. PARTICIPANTS: Fifty-four volunteers. INTERVENTION: 24-gauge angiocatheters were placed subcutaneously in both upper arms. Each arm received rHuPH20 (150 U, 750 U, or 1500 U) or equal volume saline placebo. Immediately, 400 mL Lactated Ringer's (LR) solution was gravity-infused from a 100 cm height. In the pilot stage, 5 subjects also received a similar intravenous infusion. MEASUREMENTS: Primary outcome was time to infuse 400 mL LR. Secondary outcomes included discomfort assessments, edema, arm circumference, time to recover to baseline arm circumference, subject and investigator global preference, and adverse events. RESULTS: rHuPH20 150 U, 750 U, and 1500 U yielded mean flow rates of 383 +/- 119 mL/hr, 518 +/- 154 mL/hr, and 494 +/- 136 mL/hr, respectively, compared to their respective placebo rates of 82 +/- 30 mL/hr, 148 +/- 57 mL/hr, and 124 +/- 50 mL/hr. rHuPH20 was well tolerated. CONCLUSIONS: In volunteers, clinically relevant fluid volumes can be rapidly delivered subcutaneously with rHuPH20 in a well-tolerated manner without a pump. These findings suggest that this method of hydration could potentially replace intravenous infusions in many clinical settings; further studies with rHuPH20, in patients, are warranted.

Subcutaneous drug delivery: a route to increased safety, patient satisfaction, and reduced costs.

The subcutaneous (SC) route of administration is generally preferred over intravenous administration because it enables at-home injection, improves quality of life, and reduces health care costs. In general, a volume of no greater than 1 to 2 mL is injected SC; however, for high-dose agents with limited solubility, such as monoclonal antibodies, larger volumes must be administered, which requires divided doses, smaller volumes, or more frequent dose administration. Therapeutics are being formulated with an enzyme, recombinant human hyaluronidase, to enhance the dispersion and absorption of SC administered therapeutics by transiently depolymerizing hyaluronan, a major component of the interstitial matrix.

The INFUSE-Morphine IIB study: use of recombinant human hyaluronidase (rHuPH20) to enhance the absorption of subcutaneous morphine in healthy volunteers.

Morphine is usually given intravenously (IV) for the treatment of moderate-to-severe pain, but subcutaneous (SC) administration is a viable alternative for parenteral delivery. The pharmacokinetics of SC morphine may be enhanced by coadministration with a hyaluronidase product. In this Phase IV, double-blind, randomized, crossover study, 18 healthy adults received a single dose of 2mg morphine SC with 150U of recombinant human hyaluronidase (rHuPH20), SC with 0.9% normal saline, or IV on three consecutive days. The primary endpoint was time to maximum plasma morphine concentration (T(max)) for SC injection with rHuPH20 vs. SC injection without rHuPH20. Safety and tolerability were assessed each study day, the day after the last injection, and 28 days after the last injection. After SC dosing, morphine mean T(max) was significantly shorter with rHuPH20 than without it. Mean maximum plasma morphine concentration (C(max)) after SC dosing was 29% greater with rHuPH20 than without rHuPH20 (P=0.023), although the extent of exposure of morphine was similar. T(max) was shortest and C(max) was highest with IV administration. For the major active metabolite of morphine, morphine-6-glucuronide, mean T(max) after SC morphine was significantly shorter with rHuPH20 than without rHuPH20 (a difference of approximately 17.5 minutes; P=0.0169). Coadministration of morphine with rHuPH20 appeared safe and well tolerated. Compared with SC morphine alone, rHuPH20 shortens morphine T(max) and raises C(max) in healthy adults, without changing the extent of exposure.

The INFUSE-Morphine study: use of recombinant human hyaluronidase (rHuPH20) to enhance the absorption of subcutaneously administered morphine in patients with advanced illness.

Morphine is often administered by the subcutaneous (SC) route when venous access is difficult to achieve. Hyaluronidase temporarily increases the permeability of SC connective tissues by degrading hyaluronan and has been shown to increase the dispersion and absorption of coadministered molecules. Therefore, hyaluronidase could enhance the pharmacokinetics of subcutaneous morphine. This Phase IIIB, double-blind, randomized, placebo-controlled crossover study compared the pharmacokinetics, safety, and tolerability of morphine administered SC with and without 150U of recombinant human hyaluronidase (rHuPH20) with those of intravenous (IV) morphine administration in 13 patients in a hospice or palliative care setting. Each patient received morphine 5mg parenterally daily for three days by a different method each day: IV, SC plus rHuPH20, and SC plus placebo (normal saline). The primary endpoint was the time to maximum plasma concentration (T(max)) for morphine. Concomitant SC administration of rHuPH20 enhanced the absorption rate of morphine compared with SC morphine with placebo, significantly reducing the mean T(max) from 13.8 to 9.2 minutes, a 33% decrease (P=0.026). The respective values for geometric mean maximum plasma concentration were 94.9 and 107.5nmol/L, a 13% increase (P=0.024), and the area under the plasma concentration vs. time curve values were 7.7 and 7.2micromol x min/L (P=0.23). Morphine plus rHuPH20 appeared to be safe and well tolerated. In patients requiring opioid analgesia, SC morphine plus rHuPH20 provides pharmacokinetic characteristics that are superior to those of SC morphine alone. These positive results warrant further studies on analgesic efficacy of morphine delivered with rHuPH20.

In vitro release of vascular endothelial growth factor from gadolinium-doped biodegradable microspheres.

A drug delivery vehicle was constructed that could be visualized noninvasively with MRI. The biodegradable polymer poly(DL-lactic-co-glycolic acid) (PLGA) was used to fabricate microspheres containing vascular endothelial growth factor (VEGF) and the MRI contrast agent gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). The microspheres were characterized in terms of size, drug and contrast agent encapsulation, and degradation rate. The PLGA microspheres had a mean diameter of 48 +/- 18 microm. The gadolinium loading was 17 +/- 3 microg/mg polymer and the VEGF loading was 163 +/- 22 ng/mg polymer. Electron microscopy revealed that the Gd was dispersed throughout the microspheres and it was confirmed that the Gd loading was sufficient to visualize the microspheres under MRI. VEGF and Gd-DTPA were released from the microspheres in vitro over a period of approximately 6 weeks in three phases: a burst, followed by a slow steady-state, then a rapid steady-state. Biodegradable Gd-doped microspheres can be effectively used to deliver drugs in a sustained manner, while being monitored noninvasively with MRI.