Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Olaf B. Kinstler is active.

Publication


Featured researches published by Olaf B. Kinstler.


Pharmaceutical Research | 1996

Characterization and stability of N-terminally PEGylated rhG-CSF.

Olaf B. Kinstler; David N. Brems; Scott Lauren; Amiee G. Paige; James B. Hamburger; Michael J. Treuheit

AbstractPurpose. The liquid stability of rhG-CSF was investigated after polyethylene glycol (PEG) with an average molecular weight of 6000 daltons was covalently attached to the N-terminal methionine residue. Methods. The conjugation methods chosen for modifying the N-terminal residue were alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography. The physical characterization methods of SDS-PAGE, endoproteinase peptide mapping, circular dichroism and in-vivo bioassay were used to test for differences between the PEG-rhG-CSF molecules. Results. Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temperatures, of these conjugated molecules indicated that the primary pathway of degradation was aggregation. Conjugation through alkylation offered the distinct advantage of decreasing, by approximately 5 times, the amount of aggregation present as compared to acylation. Conclusions. We suggest, that the increased stability observed for the molecules utilizing the alkylation conjugation method may be due to the preservation of charge on the alpha amino group of rhG-CSF.


Advanced Drug Delivery Reviews | 2002

Mono-N-terminal poly(ethylene glycol)–protein conjugates

Olaf B. Kinstler; Graham Molineux; Michael J. Treuheit; David Ladd; Colin V. Gegg

A site-directed method of joining proteins to poly(ethylene glycol) is presented which allows for the preparation of essentially homogeneous PEG-protein derivatives with a single PEG chain conjugated to the amine terminus of the protein. This selectivity is achieved by conducting the reductive alkylation of proteins with PEG-aldehydes at lower pH. Working examples demonstrating the application of this method to improve the delivery characteristics and therapeutic value of several proteins are provided.


Experimental Hematology | 1999

A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans

Graham Molineux; Olaf B. Kinstler; Bob Briddell; Cynthia Hartley; Patricia McElroy; Patrick Kerzic; Weston Sutherland; Greg Stoney; Brent Kern; Frederick A. Fletcher; Art Cohen; Elliot Korach; Tom Ulich; Ian K. McNiece; Pamela Lockbaum; Mary Ann Miller-Messana; Sheila Gardner; Thomas Hunt; Gisela Schwab

Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.


Protein Science | 2006

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Rahul S. Rajan; Tiansheng Li; Mohini Aras; Christopher Sloey; Weston Sutherland; Hiromi Arai; Robert Briddell; Olaf B. Kinstler; Alexis Lueras; Yu Zhang; Heather Yeghnazar; Michael J. Treuheit; David N. Brems

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N‐terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG‐GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG‐GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG‐GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG‐GCSF and 20kPEG‐GCSF. It was observed that even 5kPEG‐GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG‐GCSF. Biological activity measurements demonstrated that both 5kPEG‐GCSF and 20kPEG‐GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.


Pharmaceutical Research | 1995

The Pulmonary Absorption of Aerosolized and Intratracheally Instilled rhG-CSF and monoPEGylated rhG-CSF

Ralph W. Niven; K. Lane Whitcomb; Linda Shaner; Anna Y. Ip; Olaf B. Kinstler

AbstractPurpose. The objective of this study was to highlight differences in the pulmonary absorption of a monoPEGylated rhG-CSF and rhG-CSF after intratracheal instillation and aerosol delivery. Methods. Male Sprague Dawley rats (250 g) were anesthetized and intratracheally instilled (IT) with protein solution or were endotracheally intubated and administered aerosol for 20 min via a Harvard small animal ventilator. A DeVilbiss “Aerosonic” nebulizer containing 5 ml of protein solution at ≈ 3 mg/ml was used to generate aerosol. The volume of protein solution deposited in the lung lobes was estimated to be ≈13 µl after delivery of Tc-99m HSA solutions. The PEGylated proteins consisted of a 6 kDa (P6) or 12 kDa PEG (PI2) linked to the N-terminus of rhG-CSF. rhG-CSF also was administered IT in buffers at pH 4 and pH 7 and in dosing volumes ranging from 100 to 400 µl. Blood samples were removed at intervals after dosing and the total white blood cell counts (WBC) were determined. Plasma was assayed for proteins by an enzyme immuno assay. Results. The plasma protein concentration v. time profiles were strikingly different for aerosol v. IT delivery. The Cmax values for rhG-CSF and P12 after aerosol delivery were greater than found after IT (Aerosol: 598 ± 135 (ng/ml) rhG-CSF; 182 ± 14 P12 v. IT: 105 ± 12 rhG-CSF; 65.9 ± 5 P12). Similarly, Tmax was reached much earlier after aerosol administration (Aerosol: 21.7 ± 4.8 (min) rhG-CSF; 168 ± 31 P12 v. IT: 100 ± 17 rhG-CSF; 310 ± 121 P12). Estimated bioavailabilities (Flung %) were significantly greater via aerosol delivery than those obtained after IT (Aerosol: 66 ± 14 rhG-CSF; 12.3 ± 1.9 P12 v. IT: 11.9 ± 1.5 rhG-CSF; 1.6 ± 0.1 P12). An increase in circulating WBC counts was induced by all proteins delivered to the lungs. The rate and extent of absorption of rhG-CSF was not influenced by the pH employed nor the instilled volume. Conclusions. Estimates of bioavailability are dependent upon the technique employed to administer drug to the lungs. Aerosol administration provides a better estimate of the systemic absorption of macromolecules.


Experimental Neurology | 2001

Pegylated Brain-Derived Neurotrophic Factor Shows Improved Distribution into the Spinal Cord and Stimulates Locomotor Activity and Morphological Changes after Injury

Daniel P. Ankeny; Dana M. McTigue; Zhen Guan; Qiao Yan; Olaf B. Kinstler; Bradford T. Stokes; Lyn B. Jakeman

The neurotrophin brain-derived neurotrophic factor (BDNF) shows promise for the treatment of central nervous system (CNS) trauma and disease. Effective delivery methods are required, however, for BDNF to be useful as a therapeutic agent. To this end, we examined the penetration of intrathecally infused N-terminal pegylated BDNF (peg-BDNF) compared to similar infusion of native BDNF after spinal cord injury (SCI). Pegylation dramatically improved delivery of BDNF to the spinal cord and induced the expression of Fos in spinal cord neurons. To test whether enhanced delivery would improve the modest effects on behavioral recovery and axonal outgrowth observed with native BDNF infusion, we assessed the efficacy of 2-week 25 microg/day peg-BDNF treatment, beginning 12-24 h (early) or 15 days (delayed) after midthoracic spinal contusion. Similar to native BDNF, early treatment with peg-BDNF accelerated the recovery of stepping in the open-field and acutely stimulated locomotor central pattern generator activity, as seen by the activation of hindlimb airstepping during either period of administration. The infusion of peg-BDNF, regardless of the timing of delivery, was related to enhanced sprouting of putative cholinergic fibers, like that observed after high dose native BDNF treatment. Despite improved delivery, however, neither axonal responses nor the extent of locomotor recovery were enhanced compared to native BDNF treatment. This suggests that alternative strategies, such as neurotrophin treatment in conjunction with cell transplantation techniques, or treatment nearer the cell bodies of target neurons might be employed in an attempt to effect significant repair after SCI.


Advanced Drug Delivery Reviews | 2003

Design of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) for chronic inflammatory diseases

Carl K. Edwards; Steven Martin; James E. Seely; Olaf B. Kinstler; Scott Buckel; Alison M. Bendele; Mary Ellen Cosenza; Ulrich Feige; Tadahiko Kohno

A recombinant C-terminal truncated form of the human soluble tumor necrosis factor receptor type I (sTNF-RI) was produced in E. coli. This soluble receptor contains the first 2.6 of the 4 domains of the intact sTNF-RI molecule. A monoPEGylated form of this molecule was produced using a 30 kD methoxyPEG aldehyde with approximately 85% selectivity for the N-terminal amino group. This molecule was shown to be less immunogenic in primates than the full length (4.0 domain) molecule or other versions of sTNF-RI which were either PEGylated at different sites or with different molecular weight PEGs. The 30 kD PEG also has a longer serum half-life to the molecule than lower molecular weight PEGs. This molecule markedly blunts the inflammatory response in a number of rheumatoid arthritis animal models. In addition, phase I/II and early phase II data in humans indicate that PEG sTNF-RI is non-immunogenic and that weekly dosing with this drug can reduce the number of tender and swollen joints in rheumatoid arthritis patients. PEG sTNF-RI has comparable American College of Rheumatology (ACR) efficacy scores as other anti-TNF molecules currently used to treat rheumatoid arthritic patients.


Pharmaceutical Research | 1995

Prolonged Circulation of Recombinant Human Granulocyte–Colony Stimulating Factor by Covalent Linkage to Albumin Through a Heterobifunctional Polyethylene Glycol

Aimee Guider Paige; K. Lane Whitcomb; Jennifer Liu; Olaf B. Kinstler

AbstractPurpose. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t1/2) of rhG-CSF. Methods. Conjugates of RSA (MW 67,000) and HSA (MW 66,000) were prepared by linking the two proteins through a heterobifunctional maleimido-carboxyl polyethylene glycol (PEG) and were tested in the rat. The conjugates were injected intravenously (IV) at the equivalent dose of 50 µg/kg of rhG-CSF, and white blood cell (WBC) counts and plasma concentrations of drug were determined. A comparison of pharmacokinetic parameters was made between rhG-CSF, the conjugates RSA-PEG-rhG-CSF and HSA-PEG-rhG-CSF, and a non-covalent mixture of rhG-CSF and HSA. Results. The albumin-rhG-CSF conjugates are eliminated more slowly from the circulation. The clearance values are reduced from 0.839 ± 0.121 ml/mm/kg for rhG-CSF to 0.172 ± 0.013 ml/min/kgfor RSA-PEG-rhG-CSF and 0.141 ± 0.005 ml/mm/kg for HSA-PEG-rhG-CSF. WBC counts increased in both absolute number and duration as compared to rhG-CSF alone. The albumin rhG-CSF conjugates had enhanced serum stability relative to free rhG-CSF. The rate of degradation of the albumin conjugates incubated in rat serum at 37°C decreased five fold. Conclusions. The results from the study show that specific conjugation of rhG-CSF to albumin decreases plasma clearance in vivo, causes increased WBC response, and increases serum stability as compared to free rhG-CSF.


Archive | 1995

N-Terminally Chemically Modified Protein Compositions and Methods

Olaf B. Kinstler; Nancy E Gabriel; Christine E Farrar; Randolph B Deprince


Blood | 1995

Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice

Martha Hokom; David L. Lacey; Olaf B. Kinstler; Esther S. Choi; Steve Kaufman; Judy Faust; Chris Rowan; Erik Dwyer; Janet L. Nichol; Timothy Grasel; John Wilson; Randy Steinbrink; Randy Ira Hecht; Dwight Winters; Thomas C. Boone; Pamela Hunt

Researchain Logo
Decentralizing Knowledge