Bivalent ligands are increasingly important therapeutic agents. Although the naturally occurring antibodies are predominant, it is becoming more common to combine different antibody fragments or even low molecular weight compounds to generate heterobivalent ligands. Such ligands exhibit markedly increased affinity (i.e. avidity) and target residence time when both pharmacophores can bind simultaneously to their target sites. This is because binding of one pharmacophore forces the second tethered one to stay close to its corresponding site. This 'forced proximity' favours its binding and rebinding (once dissociated) to that site. However, rebinding will also take place when the diffusion of freshly dissociated ligands is merely slowed down. The present differential equation-based simulations explore the way both situations affect ligand binding. Both delay the attainment of binding equilibrium (resulting in steep saturation curves) and also increase the target residence time. Competitive ligands are able to interfere in a concentration-dependent manner, although much higher concentrations are required in the 'forced proximity' situation. Also, it is only in that situation that the ligand shows increased affinity. These simulations shed light on two practical consequences. Depending on the pharmacokinetic half-life of the bivalent ligand in the body, it may not have sufficient time to achieve equilibrium with the target. This will result in lower potency than expected, although it would have significant advantages in terms of residence time. In in vitro experiments, the manifestation of steep saturation curves and of accelerated dissociation in the presence of competitive ligands could mistakenly be interpreted as evidence for non-competitive, allosteric interactions. Abbreviations2D, 3D, two-and three-dimensional; (a and c), aa, ab, monovalent, homobivalent and heterobivalent ligands; A, B, target sites (ligand-bound species are named in Schemes 1 and 2); [L], f, local concentration of unbound pharmacophore of ab, handicap factor for its binding; r, maximal distance between a and b in ab

An increasing number of examples in the literature suggest that the in vivo duration of drug action not only depends on macroscopic pharmacokinetic properties like plasma half-life and the time needed to equilibrate between the plasma and the effect compartments, but is also influenced by long-lasting target binding and rebinding. The present review combines information from different research areas and simulations to explore the nature of these mechanisms and the conditions in which they are most prevalent. Simulations reveal that these latter phenomena become especially influential when there is no longer sufficient free drug around to maintain high levels of receptor occupancy. There is not always a direct link between slow dissociation and long-lasting in vivo target protection, as the rate of free drug elimination from the effect compartment is also a key influencing factor. Local phenomena that hinder the diffusion of free drug molecules away from their target may allow them to consecutively bind to the same target and/or targets nearby (denoted as 'rebinding') even when their concentration in the bulk phase has already dropped to insignificant levels. The micro-anatomic properties of many effect compartments are likely to intensify this phenomenon. By mimicking the complexity of tissues, intact cells offer the opportunity to investigate both mechanisms under the same, physiologically relevant conditions. Abbreviations2D and 3D, two-dimensional and three-dimensional; BSA, bovine serum albumin; Emax, maximal effect; GPCR, G protein coupled receptor; KD, equilibrium dissociation constant for bimolecular drug-target binding; kf, effective forward rate coefficient; koff, first-order dissociation rate constant of drug-target complex; kon, second-order association rate constant of drug-target complex; kr, effective reverse rate coefficient; PD, pharmacodynamics; PET, positron emission tomography; PK, pharmacokinetics; SPECT, Single photon emission computed tomography; t, residence time of drug-target complex; t1/2, half-life IntroductionThere are increasing examples in the literature illustrating that the long-lasting clinical action of drugs not only depends on their macroscopic pharmacokinetic properties like their plasma half-life and the time needed to equilibrate between the plasma and the effect compartments, but also on their ability to bring about long-lasting target binding. Less known but equally important are local phenomena that cause the drug molecules to accumulate near the target and/or hinder their free three-dimensional (3D) diffusion away from that target. Such hindrance may allow the same drug molecule to consecutively bind to the same target and/or targets nearby even when the free drug concentration further away has already dropped to insignificant levels. Information about the pharmacodynamic and local pharmacokinetic mechanisms that may contribute to long-lasting drug action is widely BJP British Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2010 488 British Journal of Ph...

1 CHO-K1 cells that were stably transfected with the gene for the human AT 1 receptor (CHO-AT 1 cells) were used for pharmacological studies of non-peptide AT 1 receptor antagonists. 2 In the presence of 10 mM LiCl, angiotensin II caused a concentration-dependent and long-lasting increase of inositol phosphates accumulation with an EC 50 of 3.4 nM. No angiotensin II responses are seen in wild-type CHO-K1 cells. 5 Preincubation with the insurmountable antagonist candesartan decreased the maximal angiotensin II induced inositol phosphate accumulation up to 94% and, concomitantly, decreased the maximal binding capacity of the cell surface receptors. These inhibitory e ects were halfmaximal for 0.6 nM candesartan and were attenuated by simultaneous preincubation with 1 mM losartan indicating a syntopic action of both antagonists. 6 Losartan caused a parallel rightward shift of the angiotensin II concentration-response curves and did not a ect the maximal binding capacity. EXP3174 (the active metabolite of losartan) and irbesartan showed a mixed-type behavior in both functional and binding studies. 7 Reversal of the inhibitory e ect was slower for candesartan as compared with EXP3174 and irbesartan and it was almost instantaneous for losartan, suggesting that the insurmountable nature of selective AT 1 receptor antagonists in functional studies was related to their long-lasting inhibition.

BACKGROUND AND PURPOSEOptimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy. 'Micro'-pharmacokinetic mechanisms like drug rebinding and partitioning into the cell membrane may constitute a potential fix. EXPERIMENTAL APPROACHSimulations were based on solving differential equations. KEY RESULTSBased on a selected range of association and dissociation rate constants, k on and k off , and rebinding potencies of the drugs as variables, their effects on the temporal in vivo occupancy profile of their targets, after one or multiple repetitive dosings, have here been simulated. CONCLUSIONS AND IMPLICATIONSMost strikingly, the simulations show that, when rebinding is also taken into account, increasing k on may produce closely the same outcome as decreasing k off when dosing is performed in accordance with the therapeutically most relevant constant [L max ]/K D ratio paradigm. Also, under certain conditions, rebinding may produce closely the same outcome as invoking slow diffusion of the drug between the plasma compartment and a target-containing 'effect' compartment. Although the present simulations should only be regarded as a 'proof of principle', these findings may help pharmacologists and medicinal chemists to devise ex vivo and in vitro binding kinetic assays that are more relevant and translatable to in vivo settings.Abbreviations k on , k off , second-and first-order rate constants for association and dissociation; k a , k e , first-order rate constants for the inflow and elimination/clearance of the drug; k eo , first-order rate constant for the drug's equilibration between the plasma and effect compartments; [L]

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