Ovine Hyaluronidase Research Articles

In Vivo Degradation of Crosslinked Hyaluronic Acid Fillers by Exogenous Hyaluronidases.

An advantage of hyaluronic acid (HA)-based fillers is reversibility. To evaluate the ability of 2 hyaluronidases to degrade 3 HA-based fillers using a novel in vivo model. Rats were injected with 3 HA fillers (HYC-24L+, VYC-20L, and RES-L) to create a projecting bolus. After 4 days, recombinant human hyaluronidase (HX) or ovine hyaluronidase (VIT) was administered at (1) varying doses (5 U, 10 U, or 30 U per 0.1 mL filler) or (2) different dilutions (10 U diluted 3-fold). The impact of tissue integration was assessed by administering 10 U/0.1 mL filler 4 weeks after filler injection. Three-dimensional images quantified projection loss over 72 hours. Complete loss of projection was achieved for all fillers with the highest HX and VIT doses; lower doses achieved less degradation. No difference in degradation was observed between HYC-24L+ and VYC-20L using HX or VIT. RES-L was slightly more degraded with 10 U VIT but not with 10 U HX. Enzyme dilution resulted in less degradation. Tissue integration did not impact the degree of degradation. This model incorporates the biological system while controlling variables including filler depth and volume and location of hyaluronidase delivery. Hyaluronic acid filler degradation by exogenous hyaluronidase was not hindered by differences among fillers.

Abstract: In Vivo Degradation of Hyaluronic Acid (HA)-Based Fillers by Exogenous Hyaluronidases

PURPOSE: One advantage of HA-based fillers is their reversibility. Although this has been demonstrated in vitro primarily with lab-grade hyaluronidase, there is a perception that some fillers may be more difficult to degrade in vivo. The purpose of this study was to evaluate in situ the ability of two commercially-available hyaluronidases to degrade HA fillers with different physicochemical properties. METHODS AND MATERIALS: Sprague Dawley rats were injected subcutaneously with two HA fillers, VYC-20L [20 mg/mL] or HYC-24L+ [24 mg/mL], to create a projecting bolus. Four days post-injection, recombinant human hyaluronidase (HX) or ovine hyaluronidase (VIT) were administered at varying dose levels (5U/0.1mL bolus, 10U/0.1mL bolus, and 30U/0.1mL bolus). 3D images were captured to quantify the loss of projection at six time points over 72 hours. Histology was performed to confirm degradation at 2 weeks post-administration. RESULTS: For both HA fillers, complete loss of projection was achieved with the highest dose of HX and VIT. More projection (i.e. less degradation) was detected with the lower doses of HX and VIT. No significant differences in the resulting projection were observed when comparing the effect of HX to VIT (at any dose level) or the degradation response of VYC-20L or HYC-24L+ to either hyaluronidase. The histology showed significant loss of filler material at 2 weeks, with minimal amounts of filler observed. CONCLUSION: The in vivo susceptibility of HA fillers to hyaluronidase-induced degradation has not been investigated previously. This novel animal model evaluated the susceptibility of fillers with different physicochemical properties to commercially-available hyaluronidases. Using an animal model allowed degradation to be evaluated while incorporating variables introduced by the biological environment (e.g. clearance, competing substrates for hyaluronidase). The results showed that the projection detected by 3D imaging was able to be reduced to non-detectable levels for both fillers. A dose-dependent response was observed, suggesting that the amount of degradation can be varied. Additionally, the same degree of degradation was observed for both commercially-available hyaluronidases and, despite differences in physicochemical properties, the same degree of degradation was achieved for both VYC-20L and HYC-24L+. These outcomes confirm that enzymatic degradation of HA by exogenous hyaluronidase is not hindered by the physicochemical properties of the fillers when evaluated in vivo.

Effect of microplasmin on the clearance of vitreous haemorrhage from an experimental model in rabbits

Microplasmin is known to alter the structure of the vitreous gel. The current experiments were designed to assess its ability to enhance clearance of an experimentally induced vitreous haemorrhage, and to compare it to ovine hyaluronidase. Twenty-five rabbits were used for this experiment, divided into five groups; groups 1-3 are microplasmin-treated eyes with 25, 75 and 125 μg, respectively. Group 4 treated with 55 IU of hyaluronidase, while group 5 was treated with normal saline (control). Eyes were injected in the mid-vitreous with 0.05 ml of autologous blood obtained from the marginal ear vein. One week later, all the groups were injected with the test solution injected in mid-vitreous as stated above. Clearance of the vitreous haemorrhage was assessed weekly indirect ophthalmoscopy for 8 weeks. Microplasmin-treated eyes showed a significant clearance of the vitreous haemorrhage in a dose-dependent fashion, where group 3 (125 μg) had the highest clearance rate in comparison with control eyes. Hyaluronidase-treated eyes showed a similar clearance rate as group 3. In addition, group 3 showed a complete posterior vitreous detachment, which did not develop in hyaluronidase-treated eyes. Microplasmin may be a useful agent to accelerate the clearance of vitreous haemorrhage.

Pharmacologic Vitreolysis in Diabetic Retinopathy

Diabetic retinopathy remains a major cause of worldwide preventable blindness. The vitreo-retinal interface plays a critical role in the pathogenesis of diabetic retinopathy. The term pharmacologic vitreolysis refers to the use of enzymes to liquefy the vitreous gel, and to induce posterior vitreous detachment (PVD). Intravitreal ovine hyaluronidase injection was effective in clearing vitreous hemorrhage. Several human case series demonstrated that intravitreal injection of autologous plasmin enzyme was a safe and effective adjunct to vitreous surgery for the treatment of diabetic macular edema and proliferative diabetic retinopathy. Recently, it was shown that intravitreal injection of plasmin enzyme without the performance of vitrectomy induced complete PVD and reduced macular thickening due to refractory diabetic macular edema.

Hyaluronidase: a review of approved formulations, indications and off-label use in chronic pain management

Importance of the field: Hyaluronidase for injection is an adjuvant that increases the absorption and dispersion of other injected drugs or fluids (hypodermoclysis); and improves absorption of radiopaque agents in subcutaneous urography. Ovine hyaluronidase is approved for the treatment of vitreous hemorrhages.Areas covered in this review: We review approved indications for injectable hyaluronidase and off-label uses as well as safety, efficacy and dosing information. We compare formulations made using animal tissue extracts versus the novel human recombinant type. Emphasis is on the human recombinant form and off-label uses in patients with chronic pain.What the reader will gain: Hyaluronidase reduces the obstacle that the interstitial matrix presents to fluid and drug transfer. It is a mucolytic enzyme derived from mammalian tissue or synthesized in vitro in pure form (rHuPH20) using recombinant technology. Hyaluronidase is used off-label in chronic pain management to facilitate removal of epidural adhesions with mechanical and/or hydrostatic forces and to treat edema.Take home message: The recently introduced rHuPH20 formulation obviates any risk of allergic reaction or prion-related illnesses. Reduction of edema by hyaluronidase and facilitation of epidural adhesioloysis may be beneficial in treating certain chronic painful conditions.

Angioedema after ovine hyaluronidase injection for treating hyaluronic acid overcorrection

Hyaluronic acid injections are becoming popular in aesthetic dermatology, and, sometimes, misplacements and very rarely adverse events have been reported. Hyaluronidase, an enzyme that hydrolyzes hyaluronic acid, is used to treat overcorrection or granulomatous reactions. Allergic reactions are well known except for how frequent they occur. This paper aims to confirm the efficacy of hyaluronidase injections to dissolve hyaluronic acid, but insists on the risk of hypersensitivity with animal-derived products. A case of angioedema due to ovine hyaluronidase is reported, and treatment is discussed. Hyaluronidase is highly effective, but skin test must be done before injection to avoid risk of angioedema and/or Quincke's edema.

Predicting Response of Vitreous Hemorrhage after Intravitreous Injection of Highly Purified Ovine Hyaluronidase (Vitrase) in Patients with Diabetes

To develop a predictive model for patients with diabetes who are most likely to have vitreous hemorrhage clearing by 3 months after a single, intravitreous injection of highly purified, preservative-free, ovine hyaluronidase (Vitrase; ISTA Pharmaceuticals, Inc., Irvine, CA). Post hoc data analysis was performed on two randomized, double-masked, placebo-controlled, phase 3 clinical trials of a single intravitreous injection of Vitrase for severe vitreous hemorrhage. Vitreous hemorrhage density was scored using a 0 to 4 vitreous hemorrhage grading scale in 12 radial segments of the fundus ("clock hours"). Reduction in total hemorrhage point score (DeltaTHPS) between baseline and 1 month after injection was analyzed as a predictor of vitreous hemorrhage outcome at 3 months. A strong predictive model was demonstrated by receiver operating characteristic (ROC) curve analysis; area under the curve (AUC) = 0.845 (P < 0.0001). The DeltaTHPS was higher in hyaluronidase-treated subjects than in saline-treated control subjects. Median DeltaTHPS was 8.0 and 6.0 in subjects treated with 55 IU (68 USP) and 75 IU (93 USP) of hyaluronidase respectively, versus 2.0 in saline control subjects (P < 0.0001). discussion. The DeltaTHPS at 1 month provides quantitative guidance for predicting the outcome of a single intravitreous ovine hyaluronidase injection in patients with diabetes and severe vitreous hemorrhage (ClinicalTrials.gov numbers, NCT00198510 and NCT00198497).

Safety Results of Two Phase III Trials of an Intravitreous Injection of Highly Purified Ovine Hyaluronidase (Vitrase®) for the Management of Vitreous Hemorrhage

To evaluate the safety of intravitreous ovine hyaluronidase for the management of vitreous hemorrhage. Two prospective, randomized, placebo-controlled, double-masked studies. Pooled efficacy data are presented in a companion article in this issue of The Journal. Subjects with vitreous hemorrhage > or = 1 month, severe at entry and best corrected visual acuity (BCVA) worse than 20/200 in the study eye were randomized to 7.5 IU, 55 IU, 75 IU ovine hyaluronidase, saline, or no injection. Assessments occurred on day 1, week 1, months 1, 2, 3, 6, and then every 6 months for as long as 32 months. Assessments included history, ocular symptoms, adverse events, BCVA, intraocular pressure, external eye examination, slit-lamp biomicroscopy, fundus examination, B-scan ultrasonography, and fundus photography. Of 1362 subjects in the safety population, 1344 received hyaluronidase or saline and 18 no treatment. Iritis was the most common ocular adverse event, occurring in 33.3%, 62.1%, 58.9%, and 62.1% of saline, 7.5 IU, 55 IU, and 75 IU-treated subjects. In eyes with more than mild iritis, a dose response was observed: 8.9%, 20.2%, 33.7%, and 39.7% of saline, 7.5 IU, 55 IU, and 75 IU-treated subjects, respectively, were noted to have moderate or severe iritis. Retinal detachments (RDs) were reported in 9.5% of study eyes: 26 (6.9%), 22 (11.1%), 35 (9.3%), and 45 (11.5%) in the saline, 7.5 IU, 55 IU, and 75 IU-treated subjects. Overall, 1.8% of study eyes had rhegmatogenous RD: 1.1%, 2.5%, 1.6%, and 2.3% of saline, 7.5, 55, and 75 IU treated subjects. Cataracts occurred similarly across treatment groups. No injection-related infectious endophthalmitis was reported. No serious safety issues were reported after a single intravitreous injection of ovine hyaluronidase. RD incidence was not statistically different between groups. Iritis manifesting as an acute self limited inflammation was the most common adverse event, occurred in a dose response fashion, but was not noted to result in a serious adverse event in any hyaluronidase treated eye.

Pooled Efficacy Results From Two Multinational Randomized Controlled Clinical Trials of a Single Intravitreous Injection of Highly Purified Ovine Hyaluronidase (Vitrase ®) for the Management of Vitreous Hemorrhage

To evaluate the efficacy of intravitreous ovine hyaluronidase for the management of vitreous hemorrhage. Two prospective, randomized, placebo-controlled, double-masked studies. Safety data are presented in a companion article in The Journal. Eligible patients with vitreous hemorrhage > or = 1 month duration; severe at entry with best corrected visual acuity (BCVA) worse than 20/200 were randomized to 55 IU or 75 IU ovine hyaluronidase or saline. Primary efficacy (clearance of hemorrhage sufficient to see the underlying pathology and completion of treatment when indicated) was measured at months 1, 2, and 3. Key secondary endpoints were: > or = 3-line improvement in BCVA; hemorrhage density reduction; and therapeutic utility assessment. The intent-to-treat population for common dose groups (55 IU, 75 IU, saline) consisted of 1125 patients. At baseline, 76.3% had diabetes, 90.4% were not able to read any letters on the eye chart, and mean hemorrhage duration was 120 days. Statistical significance was reached in the 55 IU dose group by months 1 and 2 for the primary efficacy endpoint based on an adjusted P-value. By months 1, 2, and 3, 13.2%, 25.5%, and 32.9% of patients (55 IU) reached primary efficacy compared with 5.5%, 16.2%, and 25.6% of saline-treated patients (P < .001; P = .002; P = .025, respectively). Key secondary endpoints confirmed the treatment effect at both doses and all timepoints (P < or = .01). Fifty-five IU ovine hyaluronidase showed statistically significant efficacy as early as months 1 and 2. These results were supported by outcomes for three key secondary endpoints. These results suggest a therapeutic utility of ovine hyaluronidase in the management of vitreous hemorrhage.

Histochemical visualization of the cartilage hyaladherins using a biotinylated hyaluronan oligosaccharide bioaffinity probe.

Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular cartilage, intervertebral disc) using a biotinylated HA (bHA) oligosaccharide bioaffinity probe. The bHA oligosaccharide probe was prepared by partial digestion of HA with ovine testicular hyaluronidase, and the oligosaccharides were labeled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaladherins were prominently visualized in tissue sections using the bHA oligosaccharide probe as pericellular components in hypertrophic epiphyseal and vertebral growth plate chondrocytes and in the enlarged cells of the cartilaginous end plate of the intervertebral disc. Weaker extracellular staining was also evident in the matrix of the ovine newborn hip and knee joint cartilages. The bHA oligosaccharide probe also visualized intracellular HABPs (IHABPs) in the hypertrophic growth plate chondrocytes of the primary ossification centers. Monolayer cultures of ovine chondrocytes rapidly internalized the bHA oligosaccharide affinity probe to discrete cytoplasmic, nuclear, and perinuclear regions, which were visualized by indirect fluorescent microscopy. This bHA oligosaccharide affinity probe may be useful in future investigations designed to characterize these novel cartilage IHABPs and the role that HA endocytosis plays in cellular regulatory processes in cartilage homeostasis.

ISTA Pharmaceuticals' NDA* for ovine hyaluronidase ['Vitrase'] has been granted priority review status

ISTA Pharmaceuticals' NDA* for ovine hyaluronidase ['Vitrase'] has been granted priority review status

Hyaluronidase (Vitrase)--ISTA: hyaluronidase--ISTA pharmaceuticals.

ISTA Pharmaceuticals (formerly Advanced Corneal Systems) has developed an ophthalmic injectable formulation of highly purified hyaluronidase [ovine hyaluronidase, Vitrase] for the initial treatment of vitreous haemorrhage and diabetic retinopathy. Hyaluronidase is a naturally occurring enzyme that digests certain forms of carbohydrate molecules called proteoglycans. The current medical treatment for vitreous haemorrhage is vitrectomy, an invasive surgical procedure that may result in future cataract formation, retinal detachment or other complications. There are currently no approved drug therapies for vitreous haemorrhage. ISTA believes that an injection of Vitrase causes the vitreous to liquefy, thereby promoting the clearance of vision-distorting blood. The elimination of blood helps to restore vision and provides an ophthalmologist with an unobstructed view of the retina, allowing the doctor to diagnose and treat the underlying cause of the hemorrhage. In mid-1997, Advanced Corneal Systems (now ISTA Pharmaceuticals) formed a Singapore subsidiary called Visionex to develop and market the company's technologies in Southeast Asia and China. In March 2000, ISTA completed the acquisition of Visionex. Also in March 2000, subsidiaries of Allergan obtained marketing, sales and distribution agreements from ISTA for Vitrase worldwide, except Mexico (until April 2004) and Japan. ISTA will split Vitrase profits equally with Allergan and receive royalties on sales in non-US countries. ISTA is responsible for all costs of product development, preclinical studies and clinical trials, of Vitrase and may receive up to 35 million US dollars in milestone payments from Allergan upon the achievement of specified regulatory and development objectives. In December 2001, Otsuka gained exclusive rights to develop, market and commercialise Vitrase in Japan. In July 2002, ISTA announced that it has entered into an agreement with Cardinal Health for the manufacture of commercial quantities of Vitrase. The agreement covers the US, Canada, Japan and the European Union. Cardinal Health will also provide manufacturing-related information for the US New Drug Application (NDA). Sophia Laboratories distribute Vitrase in Mexico. The US FDA designated Vitrase as a fast track product in October 1998, which means the FDA will facilitate the development and expedite the review of the product. Vitrase has being investigated in two multinational, randomised, placebo-controlled, phase III trials in patients with severe vitreous haemorrhage. One was conducted in the US, Mexico and Canada (North American trial) with an enrolment of 750 patients. The second trial was conducted in Europe, Brazil, Australia and South Africa and enrolled 556 patients. In March 2002, ISTA began unmasking the data, revealing that although preliminary efficacy results did not show any statistically significant improvement in the primary endpoint, clinically relevant improvements in visual acuity and a decrease in the density of vitreous haemorrhage were observed in patients treated with a 55IU dose of Vitrase, compared with placebo-treated patients. In December 2002, the FDA accepted the NDA for Vitrase for filing. The FDA's Dermatologic and Ophthalmic Drugs Advisory Committee reviewed the Vitrase NDA on 17 March 2003 and voted 8 to 4 that there was insufficient statistical evidence to support the use of Vitrasefor the treatment of vitreous haemorrhage. However, the Committee did recognise that in certain patient subgroups, the benefits of Vitrase therapy outweighed the potential risks. The FDA has recommended that ISTA provide additional analyses from the two pivotal phase III trials conducted. In April 2003, the FDA issued an approvable letter for Vitrase for the treatment of vitreous haemorrhage. ISTA anticipates that the FDA will complete its review of the Vitrase NDA anete its review of the Vitrase NDA and issue the results during the second half of 2003. In addition, ISTA plans to submit a marketing approval application with the European Medical Evaluation Agency (EMEA) in the first half of 2003. A phase II trial in Singapore was being conducted by Visionex. However, in March 2000, ISTA completed the acquisition of Visionex. In its Securities and Exchange Commission (SEC) filing, as at 31 December 2002, ISTA stated that the continued development of Vitrase for diabetic retinopathy will be dependent upon a number of factors including the FDA's evaluation of Vitrase for the treatment of vitreous hemorrhage, the successful completion of any additional clinical trials for the diabetic retinopathy, and the continuing assessment of the market opportunity for this indication compared with other product opportunities that ISTA may be pursuing at the time. ISTA is also developing hyaluronidase products for the treatment of cataracts (Keratase) and keratoconus (Keraform).

Catalytic properties of testicular hyaluronidase after gamma-irradiation.

The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.

Biochemical properties of anti-inflammatory drugs—VII. Inhibition of proteolytic enzymes in connective tissue by chloroquine (resochin) and related antimalarial/antirheumatic drugs

A chondromucoprotease present in bovine cartilage, with optimal activity at pH 5, has been partially characterized. This autolytic enzyme was irreversibly inhibited by chloroquine, those of its metabolites with a quinoline nucleus, and mepacrine (quinacrine, atebrine). These drugs did not affect cartilage degradation by a mucopolysaccharase (ovine hyaluronidase) or papain. At relatively high concentrations (10 mM), chloroquine and some of its metabolites inhibited rat skin collagenase, a presumed collagenase in bovine cartilage and a bacterial collagenase (clostridiopeptidase A). This drug action was reversible and non-competitive with the substrate, when this was gelatine. Rivanol and acetyl ammonium salts (0·25 mM) were powerful inhibitors of clostridiopeptidase A. Other types of anti-inflammatory (antirheumatic) drugs did not inhibit either the chondromucoprotease or the collagenases. A neutral sulphydryl-dependent esterase in bovine nasal cartilage was inhibited high levels of chloroquine. Chloroquine and hydroxychloroquine were metabolised (de-ethylated) by bovine cartilage incubated at pH 7·4. The possible value of (a) chloroquine and mepacrine for treating degenerative arthritis, and of (b) cetyl ammonium salts for treating clostridial infections, is discussed.