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Reid R.,ProLynx | Sgobba M.,University of California at San Francisco | Sgobba M.,University of Modena and Reggio Emilia | Raveh B.,University of California at San Francisco | And 3 more authors.
Macromolecules | Year: 2015

We have recently reported drug-releasing, degradable Tetra-PEG hydrogels as a new drug delivery system. The gels contain two self-cleaving β-eliminative linkers: one that covalently tethers the drug to the gel and releases it at a predictable rate, and another with slower cleavage that is installed in each cross-link of the polymer to control gel degradation. By balancing the two cleavage rates, the system can be designed to discharge most or all of the drug before the gel undergoes significant degradation. If polymer degradation is too rapid, undesirable gel-fragments covalently bound to the drug are released; if too slow, the gel remains in the body as an inert substance for prolonged periods. Here, we describe an analytical theory as well as a Monte Carlo simulation of concurrent drug release from and degradation of Tetra-PEG polymers. Considerations are made for an ideal network as well as networks containing missing bonds and double link defects. The analytical and simulation approaches are in perfect agreement with each other and with experimental data in the regime of interest. Using these models, we are able to (a) compute the time courses of drug release and gel degradation as well as the amount of fragment-drug conjugate present at any time and (b) estimate the rate constants of drug release and gel degradation necessary to control each of the above. We can also account for the size-dependent elimination of gel fragments from a localized semipermeable compartment and hence estimate fragment mass vs time curves in such in vivo compartments. The models described allow design of an optimal Tetra-PEG drug delivery vehicle for a particular use. © 2015 American Chemical Society.


Czudnochowski N.,University of California at San Francisco | Ashley G.W.,ProLynx | Santi D.V.,ProLynx | Alian A.,Technion - Israel Institute of Technology | And 2 more authors.
Nucleic Acids Research | Year: 2014

RluB catalyses the modification of U2605 to pseudouridine (Ψ) in a stem-loop at the peptidyl transferase center of Escherichia coli 23S rRNA. The homolog RluF is specific to the adjacent nucleotide in the stem, U2604. The 1.3 Å resolution crystal structure of the complex between the catalytic domain of RluB and the isolated substrate stem-loop, in which the target uridine is substituted by 5-fluorouridine (5-FU), reveals a covalent bond between the isomerized target base and tyrosine 140. The structure is compared with the catalytic domain alone determined at 2.5 Å resolution. The RluB-bound stem-loop has essentially the same secondary structure as in the ribosome, with a bulge at A2602, but with 5-FU2605 flipped into the active site. We showed earlier that RluF induced a frame-shift of the RNA, moving A2602 into the stem and translating its target, U2604, into the active site. A hydrogen-bonding network stabilizes the bulge in the RluB-RNA but is not conserved in RluF and so RluF cannot stabilize the bulge. On the basis of the covalent bond between enzyme and isomerized 5-FU we propose a Michael addition mechanism for pseudouridine formation that is consistent with all experimental data. © 2013 The Author(s) 2013. Published by Oxford University Press.


Henise J.,ProLynx | Hearn B.R.,ProLynx | Ashley G.W.,ProLynx | Santi D.V.,ProLynx
Bioconjugate Chemistry | Year: 2015

We have developed an approach to prepare drug-releasing Tetra-PEG hydrogels with exactly four cross-links per monomer. The gels contain two cleavable β-eliminative linkers: one for drug attachment that releases the drug at a predictable rate, and one with a longer half-life placed in each cross-link to control biodegradation. Thus, the system can be optimized to release the drug before significant gel degradation occurs. The synthetic approach involves placing a heterobifunctional connector at each end of a four-arm PEG prepolymer; four unique end-groups of the resultant eight-arm prepolymer are used to tether a linker-drug, and the other four are used for polymerization with a second four-arm PEG. Three different orthogonal reactions that form stable triazoles, diazines, or oximes have been used for tethering the drug to the PEG and for cross-linking the polymer. Three formats for preparing hydrogel-drug conjugates are described that either polymerize preformed PEG-drug conjugates or attach the drug postpolymerization. Degradation of drug-containing hydrogels proceeds as expected for homogeneous Tetra-PEG gels with minimal degradation occurring in early phases and sharp, predictable reverse gelation times. The minimal early degradation allows design of gels that show almost complete drug release before significant gel-drug fragments are released. © 2015 American Chemical Society.


Santi D.V.,ProLynx | Schneider E.L.,ProLynx | Ashley G.W.,ProLynx
Journal of Medicinal Chemistry | Year: 2014

We have recently reported a chemical approach for half-life extension that utilizes β-eliminative linkers to attach amine-containing drugs or prodrugs to macromolecules. The linkers release free drug or prodrug over periods ranging from a few hours to over 1 year. We adapted these linkers for use with phenol-containing drugs. Here, we prepared PEG conjugates of the irinotecan (CPT-11) active metabolite SN-38 via a phenyl ether that release the drug with predictable long half-lives. Pharmacokinetic studies in the rat indicate that, in contrast to other SN-38 prodrugs, the slowly released SN-38 shows a very low Cmax, is kept above target concentrations for extended periods, and forms very little SN-38 glucuronide (the precursor of enterotoxic SN-38). The low SN-38 glucuronide is attributed to low hepatic uptake of SN-38. These macromolecular prodrugs have unique pharmacokinetic profiles that may translate to less intestinal toxicity and interpatient variability than the SN-38 prodrugs thus far studied. © 2014 American Chemical Society.


Schneider E.L.,ProLynx | Henise J.,ProLynx | Reid R.,ProLynx | Ashley G.W.,ProLynx | Santi D.V.,ProLynx
Bioconjugate Chemistry | Year: 2016

We have developed a unique long-acting drug-delivery system for the GLP-1 agonist exenatide. The peptide was covalently attached to Tetra-PEG hydrogel microspheres by a cleavable β-eliminative linker; upon s.c. injection, the exenatide is slowly released at a rate dictated by the linker. A second β-eliminative linker with a slower cleavage rate was incorporated in polymer cross-links to trigger gel degradation after drug release. The uniform 40 μm microspheres were fabricated using a flow-focusing microfluidic device and in situ polymerization within droplets. The exenatide-laden microspheres were injected subcutaneously into the rat, and serum exenatide measured over a one-month period. Pharmacokinetic analysis showed a t1/2,β of released exenatide of about 7 days which represents over a 300-fold half-life extension in the rat and exceeds the half-life of any currently approved long-acting GLP-1 agonist. Hydrogel-exenatide conjugates gave an excellent Level A in vitro-in vivo correlation of release rates of the peptide from the gel, and indicated that exenatide release was 3-fold faster in vivo than in vitro. Pharmacokinetic simulations indicate that the hydrogel-exenatide microspheres should support weekly or biweekly subcutaneous dosing in humans. The rare ability to modify in vivo pharmacokinetics by the chemical nature of the linker indicates that an even longer acting exenatide is feasible. © 2016 American Chemical Society.

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