Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates

Abstract

Conjugation to macromolecular carriers is a proven strategy for improving the pharmacokinetics of drugs, with many stable polyethylene glycol conjugates having reached the market. Stable conjugates suffer several limitations: loss of drug potency due to conjugation, confining the drug to the extracellular space, and the requirement for a circulating conjugate. Current research is directed toward overcoming such limitations through releasable conjugates in which the drug is covalently linked to the carrier through a cleavable linker. Satisfactory linkers that provide predictable cleavage rates tunable over a wide time range that are useful for both circulating and noncirculating conjugates are not yet available. We describe such conjugation linkers on the basis of a nonenzymatic β-elimination reaction with preprogrammed, highly tunable cleavage rates. A set of modular linkers is described that bears a succinimidyl carbonate group for attachment to an amine-containing drug or prodrug, an azido group for conjugation to the carrier, and a tunable modulator that controls the rate of β-eliminative cleavage. The linkers provide predictable, tunable release rates of ligands from macromolecular conjugates both in vitro and in vivo, with half-lives spanning from a range of hours to >1 y at physiological pH. A circulating PEG conjugate achieved a 56-fold half-life extension of the 39-aa peptide exenatide in rats, and a noncirculating s.c. hydrogel conjugate achieved a 150-fold extension. Using slow-cleaving linkers, the latter may provide a generic format for once-a-month dosage forms of potent drugs. The releasable linkers provide additional benefits that include lowering C(max) and pharmacokinetic coordination of drug combinations.

Fig. 1.

Model and simulation of releasable PEG–drug conjugate pharmacokinetics. (Upper) One-compartment model of the fate of PEG–drug conjugates with releasable linkers. (Lower) Simulation of log concentration vs. time for conjugate (- - -), released drug (––), drug administered as bolus (….), and stable PEG conjugate (- – -). Parameters used in the simulation were k₁ = 0.0277 h⁻¹ (t_1/2, 25 h), k₂ = 0.693 h⁻¹ (t_1/2, 1 h) and k₃ = 0.0144 h⁻¹ (t_1/2, 48 h).

Fig. 2.

Pharmacokinetics of releasable PEG-AAF conjugates with substituted sulfone modulators. (A) Concentration vs. time plots of conjugates with different modulators in serum after i.v. administration to rats. The points are averages of duplicate determinations with an average SD of ±7%. (B) First-order rate constants of linker cleavage (k₁) obtained by subtraction of the terminal elimination rate of stable conjugate a (k₃) from those of cleavable linkers (k₁ + k₃) in A. (C) Tabulation of modulators and half-lives for in vivo conjugate clearance (k₁ + k₃) and cleavage (k₁) in rats, coded by letters a–h to data shown in A, B, and D. (D) Hammett plot of in vitro (–●–) and in vivo (- -■- -, rats; - -□- -, mice) PEG_40kDa-linker–AAF conjugate cleavage rates vs. σ constants of substituents on PhSO₂− modulators; σ-value of MeSO₂− modulator b is not available.

Fig. 3.

Pharmacokinetics of releasable PEG–exenatide and hydrogel PEGA–exenatide conjugates in rat. (A) PEG–exenatide conjugate with PhSO₂− modulator administered i.v. at 3 mg/kg. PEG–exenatide conjugate, ^{. .}◆^{. .}, R² = 0.96; sum of conjugate and alkenylsulfone coproduct, - -●- -, R² = 0.77; released free exenatide, –■–, R² = 0.90; and simulated curve of exenatide injected as a bolus, - – -. Points are average of duplicate determinations with an average SD of 7% for the conjugate, 6% for total PEG, and 11% for exenatide, and lines are fitted by simplex optimization of Eqs. 1 and 2 to give the best-fit values: k₁= 0.0089 h⁻¹, k₂ = 1.1 h⁻¹, k₃ = 0.015 h⁻¹, C_0,conj = 23 μM, V_D = 0.22 L/kg; reported values for k₂ and V_SS are 1.4 h⁻¹ and 0.2 L/kg, respectively (21). (B) Serum exenatide after s.c. implantation of PEGA–exenatide with pClPhSO₂− modulator at ∼8 mg/kg. Lines are fitted to data points by simplex optimization to give the best-fit values (R² = 0.91): k_abs = 0.16 h⁻¹, k₁ = 0.0089 h⁻¹, k₂ = 1.3 h⁻¹.