Medical brain notes 37

  Recombinant Human Deoxyribonuclease I INTRODUCTION   Human deoxyribonuclease I (DNase I) is an endonu-clease that catalyzes the hydrolysis of extracellular DNA. It is the most extensively studied member of a family of DNase I-like nucleases (Lazarus, 2002; Baranovskii et al., 2004; Shiokawa and Tanuma, 2001); the homologous bovine DNase I has received even greater attention historically (Laskowski, 1971; Moore, 1981; Chen and Liao, 2006). Mammalian DNases have been broadly divided into several families initially based upon their products, pH optima and divalent metal ion requirements. These include the neutral DNase I family (EC 3.1.21.1), the acidic DNase II family (EC 3.1.22.1), as well as apoptotic nucleases such as DFF40/CAD and endonuclease G (Lazarus, 2002; Evans and Aguilera, 2003; Widlak and Garrard, 2005). The human DNase I gene resides on chromosome 16p13.3 and contains 10 exons and 9 introns, which span 15 kb of genomic DNA (Kominato et al. 2006). DNase I is synth...

  PROTEIN CHEMISTRY, ENZYMOLOGY, AND STRUCTURE   The protein chemistry of human DNases including DNase I, has been recently reviewed by Lazarus (2002) and Baranovskii et al. (2004). Recombinant human DNase I is a monomeric, 260-amino acid glycoprotein (Fig. 2) produced by mammalian CHO cells (Shak et al. 1990). The protein has four cysteines, which are oxidized into two disulfides between Cys101-Cys104 and Cys173-Cys209 as well as two potential N-linked glycosylation sites at Asn18 and Asn106 (Fig. 2). rhDNase I is glycosylated at both sites and migrates as a broad band on polyacrylamide gel residues (Glu78 and Asp 212), all of which are critical for the general acid–base catalysis of phosphodiester bonds since alanine substitution of any of these results in a total loss of activity (Ulmer et al., 1996). The two Ca 2 þ  binding sites require acidic or polar residues for coordination of Ca 2 þ ; for site 1 these include Asp201 and Thr203 and for site 2 these include Asp99,...

  PHARMACOLOGY   In Vitro Activity in CF Sputum   In vitro, rhDNase I hydrolyzes the DNA in sputum of CF patients and reduces sputum viscoelasticity (Shak et al., 1990). Effects of rhDNase I were initially examined using a relatively crude “pourability” assay. Pourability was assessed qualitatively by inverting the tubes and observing the movement of sputum after a tap on the side of the tube. Catalytic amounts of rhDNase I (50  µ g/mL) greatly reduced the viscosity of the sputum, rapidly transforming it from a viscous gel to a flowing liquid. More than 50% of the sputum moved down the tube within 15 minutes of incuba-tion, and all the sputum moved freely down the tube within 30 minutes. The qualitative results of the pourability assay were confirmed by quantitativemeasurement of viscosity using a Brookfield Cone-Plate viscometer (Fig. 4). The reduction of viscosity by rhDNase I is rhDNase I concentration-dependent and is associated with reduction in size of sputum D...

  In Vivo Activity in CF Sputum   In vivo confirmation of the proposed mechanism of action for rhDNase I has been obtained from direct characterization of apparent DNA size (Fig. 7) and measurements of enzymatic and immunoreactive (ELISA) activity of rhDNase I (Fig. 8) in sputum from CF patients (Sinicropi et al., 1994a). Sputum samples were obtained one to six hours post-dose from adult CF patients after inhalation of 5 to 20 mg of rhDNase I. rhDNase I therapy produced a sustained reduction in DNA size in recovered sputum (Fig. 7), in good agreement with the in vitro data.   Inhalation of the therapeutic dose of rhDNase I produced sputum levels of rhDNase I which have been shown to be effective in vitro (Fig. 8) (Shak, 1995). The recovered rhDNase I was also enzymatically active. Enzymatic activity was directly correlated with rhDNase I concentrations in the sputum. Viscoelasticity was reduced in the recovered sputum, as well. Furthermore, results from scintigraphic stud...

  Pharmacokinetics and Metabolism   Non-clinical pharmacokinetic data in rats and monkeys suggest minimal systemic absorption of rhDNase I following aerosol inhalation of clinically-equivalent doses. rhDNase I is cleared from the systemic circulation without any accumulation in tissues following acute exposure (Green, 1994). Additionally, non-clinical me-tabolism studies suggest that the low rhDNase I concentrations present in serum following inhalation will be bound to binding proteins (Green, 1994; Mohler et al., 1993). The low concentrations of endogenous DNase I normally present in serum and the low concentrations of rhDNase I in serum following inhala-tion are inactive due to the ionic composition and presence of binding proteins in serum (Prince, 1998).   When 2.5 mg of rhDNase I was administered twice daily by inhalation to 18 CF patients, mean sputum concentrations of 2  µ g/mL DNase I were measurable within 15 minutes after the first dose on Day 1 (Fig. 9). ...

  PROTEIN MANUFACTURING AND FORMULATION   rhDNase I is expressed in mammalian cell culture and purified to homogeneity using a variety of chromato-graphic steps. The development of the formulation of rhDNase I is especially important in that a suitable formulation is required to take into account protein stability, aerosolization properties, tonicity and the sealed container for storage (Shire, 1996). rhDNase I (Pulmozyme dornase alpha) is manufactured by Genentech, Inc. and formulated as a sterile, clear, colorless aqueous solution containing 1.0 mg/mL dor-nase alpha, 0.15 mg/mL calcium chloride dihydrate and 8.77 mg/mL sodium chloride. The solution contains no preservative and has a nominal pH of 6.3. Pulmozyme is administered by the inhalation of an aerosol mist produced by a compressed air-driven nebulizer system. Pulmozyme is supplied as single-use ampoules, which deliver 2.5 mL of solution to the nebulizer.   The choice of formulation components was determined by a ...

  DRUG DELIVERY   The droplet or particle size of an aerosol is a critical factor in defining the site of deposition of the drug in the patient’s airways (Gonda, 1990). A distribution of particle or droplet size of 1 to 6  µ m was determined to be optimal for the uniform deposition of rhDNase I in the airways (Cipolla et al., 1994). Jet nebulizers have been used since they are the simplest method of producing aerosols in the desired respirable range. However, recirculation of protein solutions under high shear rates in the nebulizer bowl can present risks to the integrity of the protein molecule. rhDNase I survived recirculation and high shear rates during the nebulization process with no apparent degrada-tion in protein quality or enzymatic activity (Cipolla et al., 1994).   Approved nebulizers produce aerosol droplets in the respirable range (1–6  µ m) with a mass median aerodynamic diameter (MMAD) of 4 to 6  µ m. The delivery of rhDNase I with a device t...

  CLINICAL USE   Indication and Clinical Dosage rhDNase I (Pulmozyme dornase alpha) is currently approved for use in CF patients, in conjunction with standard therapies, to reduce the frequency of respiratory infections requiring parenteral antibiotics and to improve pulmonary function (Fig. 1). The recommended dose for use in most CF patients is one 2.5 mg dose inhaled daily using a tested, recom-mended nebulizer.       Cystic Fibrosis   rhDNase I has been evaluated in a large, randomized, placebo-controlled trial of clinically stable CF patients, 5 years of age or older, with baseline forced vital capacity (FVC) greater than or equal to 40% of predicted (Fuchs et al., 1994). All patients received additional standard therapies for CF. Patients were treated with placebo or 2.5 mg of rhDNase I once or twice a day for six months. When compared to placebo, both once daily and twice daily doses of rhDNase I resulted in a 28% to 37% reduction in respiratory trac...