DRUG DELIVERY – Hyaluronic Acid: An Ideal Ingredient for Slow-Release Formulations for Osteoarthritis Treatment


Osteoarthritis (OA) is the most common degenerative joint disorder, affecting more than 60% of the world’s population over the age of 65.1 Characterized by symptoms such as subchondral bone sclerosis, progressive articular cartilage loss, and synovial fluid viscosity decrease, OA causes bone surfaces to come into contact under ordinary load and can lead to severe pain and disability. The total economic burden of arthritis has been estimated to be 2.5% of the gross national product of western nations, and OA accounts for a large portion. In the United States alone, the costs have been estimated to be more than $60 billion per year.2

Although current medical therapies reduce the symptoms of OA, no disease-modifying drugs are approved for its treatment. For moderate-to-advanced OA, two types of local intra-articular (IA) treatments are available. The first approach is treatment with intra-articular injected anti-inflammatory drugs, which have been shown to relieve pain and other symptoms of cartilage degradation. Injections of steroids into joints and into the synovial sac have a local antiinflammator effect, and systemic side effects can often be avoided. A second strategy has been to inject hyaluronic acid (HA) preparations to restore the viscoelastic properties of the synovial fluid and increase the lubricant properties of the joint. By doing this, symptomatic pain can be reduced and joint functionality significantly improved. In addition, exogenously administered HA can reduce the production of pro-inflammatory cytokines, yielding a longer effect than the actual residence time in the joint.1

In addition to its physiological and biological effects in the joints, HA exhibits significant structural and rheological properties that make it an attractive carrier for drug delivery applications. HA can act as a depot matrix, where it slowly releases the active substance locally and, as a result, prolongs the therapeutic period of the active pharmaceutical ingredient (API) in the formulation. By adding HA into a formulation with steroid drugs, therapeutic effects can be achieved, potentially leading to more effective treatment of symptoms.


Commercially available HAs traditionally used in OA applications are produced from rooster comb extraction or various attenuated strains of Streptococcus bacteria, which have the potential to pose contamination risks from animal proteins, endotoxins, or viruses. Due to their secretion of toxins and resulting haemolytic properties, Streptococci are inherently pathogenic to human beings. This is a particular concern for regulatory bodies, such as the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). In addition, both extracted HA and Streptococcus-based HA are purified using harsh organic solvents which pose further health issues to patients.

Until recently, the source of a specific HA product has not been considered to be a clinically important point of differentiation amongst competitive products available to the market. However, with its increased use in medicinal treatments and the regulatory spotlight on potential contamination risks, demand is growing for a safer alternative to current commercial sources of HA.


In response to growing concerns across the industry toward the use of animalderived ingredients, Novozymes Biopharma has introduced Hyasis®, the next generation of high-quality hyaluronic acid, which has been developed using a Bacillus subtilis fermentation process to improve safety and minimize risks to patients. As a nonpathogenic host, Bacillus subtilis’ products are Generally Recognized as Safe (GRAS) by the FDA. The process uses minimal medium, no animal-derived raw materials, and a water-based technique, which removes the use of organic solvents at any stage during the manufacturing process. Hyasis is characterized by low amounts of nucleic acids, proteins, bacterial endotoxins, exotoxins, and microbial contamination, which reduces hypersensitivity reactions when the material is injected. It also confers a number of disease-modifying properties, including analgesic, anti-inflammatory, and chondroprotective properties. The development of the Bacillus-derived production process means that Hyasis has a reproducible molecular weight and narrow size distribution. In addition, this HA offers improved processability due to the porosity and reduced size of its spraydried particles and can dissolve much faster than HAs of streptococcal origin. This reduces filtration time at large scale and as a result, reduces manufacturing costs. A high degree of purity of the material also permits sterilization by autoclaving without significant loss of product viscosity. A recent study was conducted to evaluate the effectiveness of HA in combination with three model APIs resembling combination products for OA treatment.


To demonstrate the efficacy of Novozymes’ Hyasis in the formulation of OA therapies, 1%, 2%, and 3% HA solutions were prepared using a molecular weight of 0.85 MDa. Three model APIs were used in the release studies: diclofenac, dexamethasone phosphate, and triamcinolone hexacetonide. The diclofenac used was the commercial injectable Voltaren® (Novartis International AG) with a diclofenac concentration of 25 mg/mL, while both the dexmethasone phosphate and triamcinolone hexacetonide were pure API. Dexamethasone phosphate was dissolved in phosphate buffered saline (PBS) pH 7.4 yielding a 4-mg/mL solution. A 10-mg/mL triamcinolone hexacetonide suspension was made with 10% propylene glycol, 10% ethanol, and 0.1% sodium lauryl sulphate (SLS) in PBS buffer pH 7.4. All three APIs were in addition prepared with 1%, 2%, and 3% HA in the formulation.

The injection forces needed to inject the HA solutions were evaluated with a texture analyzer. Syringes containing HA samples were placed in the texture analyzer and exposed to constant injection speeds of 4 and 6 mL/min. This is equivalent to an injection of 1 mL over a period of 10 to 15 seconds. The average force needed to inject the material at 4 and 6 mL/min was measured, and 22G 1″ needle sizes were tested.

The release of API from the HA formulations was assessed by dissolution method using a closed loop system configuration and a USP apparatus 4 equipped with 22.6-mm cells at 37°C. Glass beads were added to fill the sample cells, and 1 mL of each formulation was added with a syringe. PBS buffer pH 7.0 was used as the medium for diclofenac and dexamethasone phosphate, while 1% SLS and 1% ethanol was added to the medium for triamcinolone hexacetonide, all equilibrated at 37°C. The flow rate was set to 4 mL/min, and a high stirring speed was used. The released API was measured online with UV absorbance. Three replicates were performed for each formulation, and a drug release above 95% was considered complete.


One of the most striking properties of HA solutions is its rheological properties and when used for treating OA, the viscous and elastic behavior of HA-based formulations is crucial.3 Novozymes has developed an HA product in which the viscoelastic properties can be altered and adjusted by changing the HA concentration, which makes it suitable for treating OA. The viscous properties of HA solutions may pose a problem for the force needed to inject the solution. In addition, flow curves show that HA solutions behave as non-Newtonian fluids with shear thinning properties. To further investigate the shear thinning effect on the ease of injection through a syringe, the force needed to inject HA solutions of different HA concentrations was measured in a texture analyzer. When Novozymes’ Hyasis was injected the injection force was constant over the entire injection period indicating a homogenous HA solution (Figure 1A). Two different injection speeds (up to 6 mL/min) were tested; all of them showing average injection forces below 6 N even at 3% HA concentrations and 6 mL/min (Figure 1B).

Dissolution testing is a critical parameter in the development of new formulations. It was initially developed for solid oral dosage forms but has in recent years expanded to other formulations, such as novel parental formulations designed as controlled-release formulations in which in vitro drug release is pivotal.4 Among the different in vitro methods available for dissolution testing, the flow through system (USP apparatus 4) offers the best characteristics for parenteral formulations and is considered a state-of-the-art method for drug release from injectable controlled- release formulations (Figure 2).5

Diclofenac (Figure 3A) is a nonsteroidal anti-inflammatory drug (NSAID) given orally, intravenously, or ocularly to reduce inflammation and pain in conditions such as arthritis and acute injury. Here, the sodium salt of diclofenac was used. The cumulative in vitro release profiles of diclofenac determined using the USP apparatus 4 method are given in Figure 3B. For the commercial injectable diclofenac formulation, a complete release was obtained after 10 minutes reflecting a reasonable water solubility and fast distribution in the dissolution system. This is the typical profile of fast-release formulations that are mostly used when administrating small molecular drugs such as diclofenac in simple formulations. By adding HA to the formulation, the release time was extended to 3, 6, and 9 hours in an HA concentration-dependent manner. This is attributed to the increased viscosity of the formulations containing HA, resulting in a slower diffusion of the API. No initial burst release of diclofenac was observed for all three HA concentrations. As a result, the diclofenac formulation containing 3% HA showed a steady release profile with a 50 times slower release compared to a formulation without HA.

Dexamethasone phosphate (Figure 4A) is a corticosteroid that has an antiinflammatory effect. The cumulative in vitro release profiles of dexamethasone phosphate determined using the USP apparatus 4 method are given in Figure 4B. Again, the API dissolved in PBS showed a fast distribution in the dissolution system, and a complete release was obtained after 10 minutes. The effect of HA in the formulation was similar to that of diclofenac, in which a complete release of dexamethasone phosphate was obtained after 9 to 10 hours when formulated with 3% HA. No burst release was observed, although the release profile of dexamethasone phosphate showed greater curvature when compared to diclofenac.

Triamcinolone hexacetonide (Figure 5A) is another corticosteroid used to treat arthritis and designed for a slow release due to its low aqueous solubility. This effect was observed when triamcinolone hexacetonide was formulated as a suspension without HA. Here, the in vitro release profile was significantly different from diclofenac and dexamethasone phosphate (Figure 5B). 80% API was released after 2 hours in an initial relatively fast-release period followed by a slower-release phase in which 95% was released after 6 hours and 100% after 12 hours. By adding HA to the formulation, the release rate in the initial phas decreased, while the rate in the second phase remained constant. Subsequently, the initiation of the second phase was shifted from 2 hours without HA to 10 hours with 3% HA in the formulation, and a 95% drug release was not achieved during the 12 hours.


As the future of OA treatments lies in the development of more effective therapies, the market will most likely see a rise in advanced and multifunctional solutions utilizing combinations of HA and APIs. Three suitable APIs were formulated with Novozymes’ Hyasis as models for future combination products. Formulating all three tested APIs with HA resulted in slower release time in an HA concentration-dependent manner. The effect of HA on diclofenac and dexamethasone phosphate release was similar, leading to 50 times slower release. For triamcinolone hexacetonide, the effect was smaller but resulted in the longest total release time of the three APIs. As a result, combining HA and an API could lead to a more effective OA treatment in which the viscoelastic properties of HA and the anti-inflammatory effect of the API are combined in a controlled-release formulation.

Bacillus-derived HA offers new possibilities to the industry by providing  pure and biocompatible source that confers numerous advantages to both practitioners and patients. The innovative technology offers unmatched ease of administration, effectiveness, and an unequalled safety profile, which make it highly suitable for drug delivery applications.


1. Gerwin N, Hops C, Lucke A. Intraarticular drug delivery in osteoarthritis. Adv Drug Deliver Rev. 2006;58:226-242.
2. Buckwalter JA, Martin JA. Osteoarthritis. Adv Drug Deliver Rev. 2006; 58:150-167.
3. Balazs EA. Therapeutic use of hyaluronan. Struct Chem. 2009;20:341- 349.
4. Siewer M, Dressman J, Brown C, Shah V. FIP/AAPS Guidelines for dissolution/in vitro release testing of novel/special dosage forms. Dissolut Technol. 2003;2:6-15.
5. Bhardwaj U, Burgess DJ. A novel USP apparatus 4 based release testing method for dispersed systems. Int J Pharm. 2010;388(1-2):287-294.

Morten J. Maltesen joined Novozymes Biopharma in 2010 as a Research Scientist. He earned his MSc in Engineering (specialized in Biotechnology) from the Technical University of Denmark combined with his PhD in Pharmaceutical Sciences from the University of Copenhagen. In his current role as project manager, Dr. Maltesen works on slow-release formulations based on recombinant hyaluronic acid. Before joining Novozymes Biopharma, he worked as a research scientist in early drug development.

Ole M. Dall is a Formulation Scientist and Pharmacist from the University of Copenhagen. He joined Novozymes Biopharma in 2009 as a Customer Technical Solution Scientist. In this role, he works with customers and partners that are using and evaluating Novozymes’ hyaluronic acid and technologies within this field. Prior to this position, Mr. Dall worked as a Formulation Pharmacist at a generic drug development company and as a Medical Consultant in the Danish pharmacy association.