The potential of in vitro pharmacokinetic profiling to predict oral bioavailability of carbohydrate mimetics and cyclic hexapeptides

Urinary tract infection (UTI) is one of the most prevalent infectious diseases and is mainly caused by uropathogenic Escherichia coli (UPEC). The first step of the infection cycle, the adhesion of UPEC to urothelial cells, is mediated by the lectin FimH, which is located at the tip of bacterial type 1 pili. Its interaction with the mannosylated glycoprotein uroplakin Ia (UPIa) on urothelial cells can be prevented by glycomimetic antagonists. Thus, alpha-D-mannopyranosides derivatives with nanomolar affinity offer a potential therapeutic approach for prevention and treatment of UTI. To achieve oral availability as well as a therapeutic concentration in the bladder over an extended period of time, a number of key issues have to be fulfilled. • Stability of the antagonist against various gastrointestinal conditions (pH, enzymatic activity) is required. • Sufficient solubility is a prerequisite for successful absorption. • To reach circulation, the intestinal mucosa has to be permeated. • In case of a prodrug approach, hepatic first pass metabolism is requested. • To reach the therapeutic target in the bladder, renal excretion is required. These various properties can be predicted based on lipophilicity (log D7.4), stability in different gastrointestinal fluids, kinetic and thermodynamic solubility, metabolic stability (liver microsomes), and permeability (parallel artificial membrane permeability assay and Caco-2 cells assay), methods implemented in the PADMET platform (physicochemical properties, absorption, distribution, metabolism, elimination and toxicity) of our research group. The aim of this thesis is to develop in vitro assays to provide information regarding the factors influencing the pharmacokinetic properties of glycomimetics and to predict their oral bioavailability. For this purpose, we developed our PADMET platform. The focus was on glycomimetic FimH antagonists and the evaluation of their structural, physicochemical, and biochemical properties. Besides in vitro assays to determine their solubility and potential passive permeability, assays predictive for pKa, stability in gastrointestinal fluids, and carrier-mediated permeability were implemented into our PADMET platform. As a result, low passive membrane permeability constraint oral absorption of mannosidic FimH antagonists with para-carboxy-biphenyl aglycone. With ester prodrugs permeability could be improved and the hydrolysis by carboxylesterase released the active principle within minutes. Unfortunately, the ester prodrugs suffer from low solubility. For early in vivo trials, the solubility issue was addressed with phosphate prodrugs or with appropriate preformulations using co-solvents, surfactants, and complexing agents. In addition, phosphate and ester prodrugs were stable in gastrointestinal fluids, whereas a prodrug with acylation in the C-6 position of the mannose moiety showed instability in intestinal fluids due to the enzyme pancreatin. Furthermore, interactions of FimH antagonists with carrier-mediated transporters were observed and require further investigations. In a collaborative project with the research group of Prof. Horst Kessler (Technical University of Munich, Institute for Advanced Study) the in vitro permeability of cyclic hexapeptides was evaluated with log D7.4, PAMPA, and Caco-2 cells. The cyclic hexapeptides were synthesized in the research group of Prof. Dr. Horst Kessler and permeability experiments were performed in our laboratory. As a result, di-N-pentyl alkylation of cyclic hexapeptides improved passive permeability. In addition, differential Caco-2 permeability was measured for enantiomers suggesting the involvement of carrier-mediated transporters

Enantiomeric cyclic peptides with different Caco-2 permeability suggest carrier-mediated transport

Recently, oral absorption of cyclic hexapeptides was improved by N-methylation of their backbone amides. However, the number and position of N-methylations or of solvent exposed NHs did not correlate to intestinal permeability, measured in a Caco-2 model. In this study, we investigate enantiomeric pairs of three polar and two lipophilic peptides to demonstrate the participation of carrier-mediated transporters. As expected, all the enantiomeric peptides exhibited identical lipophilicity (logD7.4) and passive transcellular permeability determined by the parallel artificial membrane permeability assay (PAMPA). However, the enantiomeric polar peptides exhibited different Caco-2 permeability (Papp) in both directions a-b and b-a. The same trend was observed for one of the lipophilic peptide, whereas the second lipophilic enantiomer pair showed identical Caco-2 permeability (within the errors). These findings provide the first evidence for the involvement of carrier-mediated transport for peptides, especially for those of polar nature

FimH antagonists - solubility vs. permeability

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are among the most prevalent infections worldwide. Since frequent antibiotic treatment favors the emergence of antibiotic resistance, efficient non-antibiotic strategies are urgently needed. The first step of the pathogenesis of UTI is the bacterial adherence to urothelial host cells, a process mediated by the mannose-binding adhesin FimH located at the tip of bacterial pili. In a preliminary study, biphenyl α-d-mannopyranosides with an electron-withdrawing carboxylate on the aglycone were identified as potent FimH antagonists. Although passive permeability could be established by masking the carboxylate as an ester, insufficient solubility and fast hydrolysis did not allow to maintain the therapeutic concentration in the bladder for the requested period of time. By modifying the substitution pattern, molecular planarity and symmetry of the biphenyl aglycone could be disrupted leading to improved solubility. In addition, when heteroatoms were introduced to the aglycone, antagonists with further improved solubility, metabolic stability as well as passive permeability were obtained. The best representative, the pyrrolylphenyl mannoside 42f exhibited therapeutic urine concentration for up to 6 h and is therefore a promising oral candidate for UTI prevention and/or treatment

FimH Antagonists: Phosphate Prodrugs Improve Oral Bioavailability

The widespread occurrence of urinary tract infections has resulted in frequent antibiotic treatment, contributing to the emergence of antimicrobial resistance. Alternative approaches are therefore required. In the initial step of colonization, FimH, a lectin located at the tip of bacterial type 1 pili, interacts with mannosylated glycoproteins on the urothelial mucosa. This initial pathogen/host interaction is efficiently antagonized by biaryl α-d-mannopyranosides. However, their poor physicochemical properties, primarily resulting from low aqueous solubility, limit their suitability as oral treatment option. Herein, we report the syntheses and pharmacokinetic evaluation of phosphate prodrugs, which show an improved aqueous solubility of up to 140-fold. In a Caco-2 cell model, supersaturated solutions of the active principle were generated through hydrolysis of the phosphate esters by brush border-associated enzymes, leading to a high concentration gradient across the cell monolayer. As a result, the in vivo application of phosphate prodrugs led to a substantially increased Cmax and prolonged availability of FimH antagonists in urine

FimH Antagonists: Bioisosteres To Improve the in Vitro and in Vivo PK/PD Profile

Urinary tract infections (UTIs), predominantly caused by uropathogenic Escherichia coli (UPEC), belong to the most prevalent infectious diseases worldwide. The attachment of UPEC to host cells is mediated by FimH, a mannose-binding adhesin at the tip of bacterial type 1 pili. To date, UTIs are mainly treated with antibiotics, leading to the ubiquitous problem of increasing resistance against most of the currently available antimicrobials. Therefore, new treatment strategies are urgently needed. Here, we describe the development of an orally available FimH antagonist. Starting from the carboxylate substituted biphenyl α-d-mannoside 9, affinity and the relevant pharmacokinetic parameters (solubility, permeability, renal excretion) were substantially improved by a bioisosteric approach. With 3'-chloro-4'-(α-d-mannopyranosyloxy)biphenyl-4-carbonitrile (10j) a FimH antagonist with an optimal in vitro PK/PD profile was identified. Orally applied, 10j was effective in a mouse model of UTI by reducing the bacterial load in the bladder by about 1000-fold