title: Synthese neuartiger Thrombinhemmstoffe zur Entwicklung gerinnungshemmender Liposomen
title: Synthesis and incorporation of Thrombin Inhibitors in to liposomes
Creators: name: Endreas, Wegderes
Publisher: name: Philipps-Universität Marburg
Year: 2016
Subjects: gerinnungshemmende Liposomen
Thrombininhibitoren
anticoagulants
Medical sciences, Medicine
Medizin, Gesundheit
Antikoagulans , Koagulation
liposomes, thrombin inhibitors
Liposom

description: Im Rahmen dieser Dissertation ist
      es gelungen ist, hochwirksame Thrombininhibitoren mit
      Palmitinsäure-, Biotin- und bifunktionellen
      Ethylenglykol-Resten zu modifizieren, wobei die Hemmwirkung
      nahezu erhalten blieb. Die freien biotinylierten Inhibitoren
      eignen sich zur Untersuchung von
      Rezeptor-Ligand-Wechselwirkungen, beispielsweise mittels
      SPR-Messungen. Die palmitoylierten Inhibitoren wurden in die
      DPPC/CH-Liposomen eingebaut. Ein damit verbundenes Ziel war
      eine Molekulargewichtsvergrößerung der Wirkstoffe, die zu
      einer deutlichen Einschränkung der renalen Filtration führen
      sollte. Obwohl die Wirkung dieser Konjugate in vivo noch
      nicht gezeigt werden konnte, ist anzunehmen, dass der
      Konjug-ationsprozess eine verlängerte Verweildauer der
      Inhibitoren in der Zirkulation zur Folge haben dürfte. Die in
      dieser Arbeit beschriebenen Methoden, insbesondere die
      Multimerisierung, lassen sich auch auf andere Wirkstoffe
      übertragen. Voraussetzung dafür ist allerdings, dass sich die
      betreffenden Wirkstoffe ohne Aktivitätsverluste über einen
      geeignenten flexiblen Spacer mit Fettsäureresten modifizieren
      lassen. Durch die infolge der Multimerisierung erzielte
      Vergrößerung des Molekulargewichtes der Wirkstoffe lässt sich
      die jeweilige renale Eliminierungsgeschwindigkeit wesentlich
      reduzieren. Folglich müssen diese Wirkstoffe weniger häufig
      verabreicht werden, um den erforderlichen Wirkstoffspiegel am
      Wirkort zu gewährleisten. Hierdurch ergeben sich in der
      Praxis relevante Vorteile wie geringere Toxizität sowie
      effizientere Kosten-Nutzen-Relationen.
description: Peptides are recognized for being
      highly selective and efficacious; simultaneously, they are
      relatively safe and well-tolerated. As a result, there is an
      increased interest in peptides in pharmaceutical research and
      development. Meanwhile, over 60 peptide-based drugs have been
      approved by the FDA and approximately 140 peptide
      therapeutics are currently being evaluated in clinical
      trials. However, many peptide and peptidomimetic drugs suffer
      from rapid clearance in vivo. This phenomenon can be reduced
      by increasing the respective size through oligomerization or
      covalent conjugation with polymers. The objective of this
      doctoral thesis was to develop a new and convenient strategy
      for drug oligomerization, in which peptidomimetic thrombin
      inhibitors are to be incorporated into the liposomes. For
      this purpose, two types of benzamidine-derived thrombin
      inhibitors were used which were initially modified with
      palmitic acid to enable their incorporation into liposomes.
      Type I inhibitors contain an L-configurated trifunctional
      amino acid (lysine) in P3 position and inhibit thrombin in
      the low nanomolar range, while possessing negligible potency
      against related proteases such as factor Xa, plasmin, uPA,
      plasma kallikrein and matriptase. The inhibitor moiety was
      covalently coupled to a palmitic acid residue, either
      directly to the P3-side chain or via a short bifunctional
      ethylene glycol spacer. The second type of inhibitors with
      D-cyclohexylalanine in P3-position is less specific and
      inhibits besides thrombin also factor Xa, although in a
      significantly weaker way. Type II inhibitors have been
      modified at their sulfonyl residue in P4 position. For both
      inhibitor-type series, strong thrombin-inhibitory potencies
      were obtained in enzyme kinetic studies, showing that the
      modification was very well-tolerated. The palmitoylated
      molecules were directly added to the lipid mixture (DPPC,
      cholin) which was used for liposome preparation. The
      inhibitory potency of the obtained liposomes was determined
      by enzyme kinetic studies. Furthermore, the physicochemical
      properties of the liposomes and their anticoagulant
      activities were characterized in plasma. The obtained
      liposomes possess strong thrombin inhibitory potency in
      enzyme kinetic measurements as well as anticoagulant activity
      in plasma. Their strong potency and positive ζ potential
      indicate that large amounts of the benzamidine-derived
      inhibitors are located on the surface of the liposomes. The
      liposomes prepared from inhibitor containing L-lysine-residue
      in P3 position, elongated with ethylene glycol linker, show
      excellent inhibitory potency (IC50 = 0.55 nM), whereas for
      the vesicles with the compound lacking the spacer a strongly
      reduced activity were determined (IC50 = 81 nM). This result
      indicates the importance of the flexible spacer between the
      inhibitor-head and the surface of the liposomes to improve
      the accessibility for the target enzyme. Moreover, employing
      a long flexible spacer would facilitate the binding of
      several thrombin molecules to inhibitor-loaded liposomes,
      resulting in enhanced inhibitory effects. In case of
      complications in the drug treatment, it is highly important
      to have a specific tool in order to reverse the action of
      drugs. In a previous study conducted by Olson etal., a
      chimeric anticoagulant containing both an indirect factor Xa
      inhibitor (fondaparinux analog) and a direct thrombin
      inhibitor (α-NAPAP analog) has been prepared. Furthermore the
      Lys-side chain of this compound was additionally coupled to
      biotin, which enables a neutralization of their anticoagulant
      activity by injecting avidin. To adapt this antidote
      strategy, bifunctionalized liposomes were prepared containing
      the inhibitor, along with the additional biotinylated
      compound Me-(CH2)14-CO-Tota-Biotin on their surface. However,
      it was only possible to achieve a partial neutralization of
      the thrombin inhibitory potency by the addition of avidin. A
      stronger neutralizing effect was observed for free
      biotinylated inhibitors. In this doctoral thesis described
      strategy could be also applied to other drug molecules which
      suffer from rapid renal clearance and tolerate modification
      with a linker coupled to a suitable fatty acid. Although we
      did not perform any pharmacokinetic studies, we assume that
      incorporation of these inhibitors in to liposomes should
      increase their half-lives in circulation relative to that of
      the unmodified peptidic drug molecules.