Multidose Hyaluronidase Administration as an Optimal Procedure to Degrade Resilient Hyaluronic Acid Soft Tissue Fillers

Abstract

Minimally invasive hyaluronan (HA) tissue fillers are routinely employed to provide tissue projection and correct age-related skin depressions. HA fillers can advantageously be degraded by hyaluronidase (HAase) administration in case of adverse events. However, clear guidelines regarding the optimal dosage and mode of administration of HAase are missing, leaving a scientific gap for practitioners in their daily practice. In this study, we implemented a novel rheological procedure to rationally evaluate soft tissue filler degradability and optimize their degradation kinetics. TEOSYAL RHA^® filler degradation kinetics in contact with HAase was monitored in real-time by rheological time sweeps. Gels were shown to degrade as a function of enzymatic activity, HA concentration, and BDDE content, with a concomitant loss of their viscoelastic properties. We further demonstrated that repeated administration of small HAase doses improved HA degradation kinetics over large single doses. Mathematical analyses were developed to evaluate the degradation potential of an enzyme. Finally, we tuned the optimal time between injections and number of enzymatic units, maximizing degradation kinetics. In this study, we have established a scientific rationale for the degradation of HA fillers by multidose HAase administration that could serve as a basis for future clinical management of adverse events.

Keywords: enzymatic degradation; hyaluronic acid; hyaluronidase; rheology; soft tissue filler.

Figure 1

Degradation of Teosyal PNT gels in contact with Hylenex or Hyalase for 180 min, assessed by rheological time sweeps (37 °C, strain 0.1%, 1 Hz). (A) Degradation of PNT gels in contact with 7.1 U·mL⁻¹ of Hylenex over a 3-h period, (B) Degradation of PNT gels in contact with 7.1 U·mL⁻¹ of Hyalase over a 3-h period, degradation rates of Hylenex-treated (C) and Hyalase-treated (D) PNT gels calculated from the first derivatives, (E) time required to reach 50% of gel degradation (half-life) of PNT gels with Hylenex or Hyalase enzymes, (F) time needed to reach an enzymatic degradation speed inferior to 1 Pa·s⁻¹, (G) example of a PNT4 gel before (left) and after (right) treatment with the Hylenex enzyme. All results are presented as mean ± S.D, n = 2.

Figure 2

Evaluation of PNT4 degradation by single or multidose injections of Hylenex or Hyalase enzymes, followed by rheological time sweeps. (A) Schematic of the method used to degrade the PNT4 gels by single or multidose enzyme injections, (B) Evolution of the storage modulus of PNT4 gels over time, measured by time sweeps (37 °C, strain 0.1%, 1 Hz) after single, double, or triple injections of the Hylenex enzyme. For all conditions, the total volume of enzyme added is 90 μL, for a total enzymatic concentration of 34.6 U·mL⁻¹; blue arrows indicate enzyme addition, (C) First derivative curves of single, double, and triple dose injections, (D) Evolution of the storage modulus of PNT4 gels over time, measured by time sweeps (37 °C, strain 0.1%, 1 Hz) after single, double, or triple injections of the Hyalase enzyme. For all conditions, the total volume of enzyme added is 60 μL, for a total number of 277.8 U·mL⁻¹; blue arrows indicate enzyme addition, (E) First derivative curves of single, double, and triple dose injections; blue arrows indicate enzyme injections with the Hyalase enzyme, (F) Crossover time at which multidose storage moduli are lower than with a single dose, (G) Storage moduli at plateau obtained after single, double, or triple addition of enzyme, (H) area under the curve (AUC) extracted from the degradation rates of PNT4 gels using both enzymes. All results are presented as mean ± S.D, n = 3.

Figure 3

Refinement of PNT4 degradation parameters by triple injections of Hylenex, followed by rheological time sweeps. (A) Evolution of the storage modulus of PNT4 gels over time, measured by time sweeps (37 °C, strain 0.1%, 1 Hz), following triple injections of the Hylenex enzyme (34.6 U·mL⁻¹) with injection spans of 5, 10, 20, and 30 min, Percentage of gel degradation after 30 (B) or 120 (C) min for each injection span, compared to the initial storage modulus set at 300 Pa, (D) Evolution of the storage modulus of PNT4 gels over time, measured by time sweeps (37 °C, strain 0.1%, 1 Hz), following triple injections of the Hylenex enzyme 20 min apart, with enzymatic unit numbers of 9.4, 20.2, 34.6, and 46.2 units of HAase per mL of gel, percentage of gel degradation after 30 (E) or 120 (F) min for each enzymatic unit number, compared to the initial storage modulus set at 300 Pa. All results are presented as mean ± S.D, n = 3. Mann–Whitney non-parametric tests were performed on (B,C,E,F), with a confidence level α = 0.05 (* p < 0.05).

Figure 4

Optimization of PNT4 and PNT3 degradation by multiple injections of 38.3 U·mL⁻¹ Hylenex every 5 min, followed by rheological time sweeps. Evolution of the storage modulus of PNT4 (A) and PNT3 (B) gels over time, measured by time sweeps (37 °C, strain 0.1%, 1 Hz) following single or multiple injections of the Hylenex enzyme (38.3 U·mL⁻¹ per injection, 5 min injection span for multidose), total volume and enzymatic units were equivalent to single and multidose. Dotted lines represent 10% of initial G’, First derivative curves of PNT4 (C) and PNT3 (D) gels for single and multidose injections, area under the curve of PNT4 (E) and PNT3 (G) gels calculated from the first derivative curves, time required to reach 90% of gel degradation for PNT4 (F) and PNT3 (H) gels, relative to the initial storage modulus. All results are presented as mean ± S.D, n = 3.