The Pharmacodynamics Study Of Recombinant Human Hyaluronidase

HA is a glycosaminoglycan that exists as a polymer of repeating disaccharides.The disaccharide is composed of N-acetyl-D-glucosamine and D-glucuronic acid,which are linked via alternating β-1,4 and β-1,3 glycosidic bonds.On account of the high degree of polymerization,natural HA exists primarily as a high molecular weight(HMW HA >2×106k Da)and wide molecular weight distribution form.Molecular weight of the HA is the important parameter of biological function.The extracellular matrix(ECM)contains plenty of HMW HA that results in high viscosity to resist compressive forces.Depend on its viscoelasticity,HMW HA has been applied in eye surgery.Depend on its lubrication and the special ability of water retention,LMW HA(1×105Da<Mr<2×106Da)has been applied in cosmetics.The research found that the VLMW HA(Mr<1×105Da)has extraordinarily wide variety of biological functions including angiogenesis,wound healing,immunity regulation and tumor therapy.Therefore,the production of high purity VLMW HA is pretty developing value.Here we perform the enzymatic hydrolysis of the HMW HA with ru HPH20.Utilize the Gel Permeation Chromatography(GPC)to measure the weight-average molecular(Mw)of the enzymatic products at the various reaction time.Base on the Michaelis-Menten equation and the Mw of the enzymatic products at the various reaction time,we establish a kinetic model: 1/tM = 0.0000000891t+0.0000080679(R2 = 0.991)(1/tM is the Mw of the enzymatic products at the various reaction time,t is reaction time).According to the kinetic model,arbitrary molecular weight of HA can be gained by controlling the reaction time.HAase are a family of enzyme that predominately degrade HA with limited ability to degrade chondroitin and chondroitin sulfates.There are three subtypes of human hyaluronidases:Hyaluronidase 1(h Hyal-1),hyaluronidase 2(h Hyal-2)and hyaluronidase PH20(PH20).PH20 is the major HAase for degrading human HA.We established a r Hu PH20 scale production process and obtained the r Hu PH20 with high purity.Subcutaneous injection therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets.The interstitial matrix of the skin is comprised of numerous structural macromolecules including elastin,collagens,fibronectin and HA that form complex three-dimensional structure substance which acts as a filter limits the type and quantity of drugs administered by local injection.HA is the “ground substance” of the structure that acts as a barrier to hinder fluid flow through the interstitial matrix and contributes to drainage of accumulated edematous fluid by way of its viscosity and water of hydration.Here we perform experiments that facilitates diffusion of locally injected drugs on nude mouse,r Hu PH20 was also injected to rabbit before subcutaneous injection of large volumes of physiological saline that turn out to be minimal distortion and tearing of the subcutaneous tissue.We found that r Hu PH20 contributes to subcutaneously injection as “Spreading agents”.r Hu PH20 temporarily reduces viscosity of HA which improves tissue diffusion of locally co-injected drugs.Importantly,the half-life of HA in the interstitial matrix of the skin is short of 15–20 h which means that the interstitial viscoelastic barriers are restored without histologic alterations or signs of inflammation within 24 h.The half-life of r Hu PH20 in vivo is short of 5 min which means that it hardly cause the damage of tissue and the inflammatory reaction.Meanwhile,r Hu PH20 is an enzyme with substrate specificity means that has no effect on the efficacy of co-injected drugs and no damage of tissue.r Hu PH20 is considered as “Spreading agents” with good compatibility and functionality applied to subcutaneously injection that ensure the drugs’ safety in clinic.Subcutaneously injection with assisted r Hu PH20 that function as a “Spreading agents” is a new “large-volume interstitial drug delivery pathway”,Co-administration of r Hu PH20 is a potential option to enhance the diffusion and infusion quantity,thereby it meets the needs of quick drug delivery and large-volume interstitial drug delivery.There are great application prospects:(1)Depend on simplicity operation and highly bioavailability,It is possible for patients with chronic diseases replace intravenously administered pharmaceuticals by self-administer injectable,such as injection of insulin by diabetic.(2)Replace intravenous delivery when the circumstances and conditions are not suitable for intravenous injection,such as burn patients.(3)Provide a new way to administrate the drugs which unable injected intravenously due to limitations on drug solubility,bioavailability or stability.(4)Meet the needs of quick drug delivery and large-volume interstitial drug delivery in the particular circumstances,such as administrate detoxification even during the solo mission in the battlefield.Intranasal r Hu PH20 combination with NGF administration proved to a suitable alternate delivery route which facilitated NGF to enter brain tissues,the delivery rate of NGF and the concentration do NGF in the brain tissues was significantly increased.There is great application prospect that treat the diseases associated with nerve system traumatic.