Formulation, stabilisation and encapsulation of bacteriophage for phage therapy

Against a backdrop of global antibiotic resistance and increasing awareness of the importance of the human microbiota, there has been resurgent interest in the potential use of bacteriophages for therapeutic purposes, known as phage therapy. A number of phage therapy phase I and II clinical trials have concluded, and shown phages don’t present significant adverse safety concerns. These clinical trials used simple phage suspensions without any formulation and phage stability was of secondary concern. Phages have a limited stability in solution, and undergo a significant drop in phage titre during processing and storage which is unacceptable if phages are to become regulated pharmaceuticals, where stable dosage and well defined pharmacokinetics and pharmacodynamics are de rigueur. Animal studies have shown that the efficacy of phage therapy outcomes depend on the phage concentration (i.e. the dose) delivered at the site of infection, and their ability to target and kill bacteria, arresting bacterial growth and clearing the infection. In addition, in vitro and animal studies have shown the importance of using phage cocktails rather than single phage preparations to achieve better therapy outcomes. The in vivo reduction of phage concentration due to interactions with host antibodies or other clearance mechanisms may necessitate repeated dosing of phages, or sustained release approaches. Modelling of phage-bacterium population dynamics reinforces these points. Surprisingly little attention has been devoted to the effect of formulation on phage therapy outcomes, given the need for phage cocktails, where each phage within a cocktail may require significantly different formulation to retain a high enough infective dose. This review firstly looks at the clinical needs and challenges (informed through a review of key animal studies evaluating phage therapy) associated with treatment of acute and chronic infections and the drivers for phage encapsulation. An important driver for formulation and encapsulation is shelf life and storage of phage to ensure reproducible dosages. Other drivers include formulation of phage for encapsulation in micro- and nanoparticles for effective delivery, encapsulation in stimuli responsive systems for triggered controlled or sustained release at the targeted site of infection. Encapsulation of phage (e.g. in liposomes) may also be used to increase the circulation time of phage for treating systemic infections, for prophylactic treatment or to treat intracellular infections. We then proceed to document approaches used in the published literature on the formulation and stabilisation of phage for storage and encapsulation of bacteriophage in micro- and nanostructured materials using freeze drying (lyophilization), spray drying, in emulsions e.g. ointments, polymeric microparticles, nanoparticles and liposomes. As phage therapy moves forward towards Phase III clinical trials, the review concludes by looking at promising new approaches for micro- and nanoencapsulation of phages and how these may address gaps in the field

Bicyclic octapeptide alpha-Amanitin, the death cap mushroom toxin : the total synthesis and derivatives of the hydroxyproline residue

This thesis presents the first total synthesis of the death cap mushroom toxin α-amanitin and the synthesis of its derivatives containing analogues of the hydroxyproline residue. In Chapter 2, an enantioselective route to the synthesis of (2S,3R,4R)-dihydroxyisoleucine, an unnatural oxidized amino acid found in α-amanitin, is presented. This includes the synthetic challenges that needed to be overcome, previous non-enantioselective syntheses of this amino acid, my failed attempts, and eventually the route to successfully obtain the desired enantiomer of this residue. Chapter 3 describes an unprecedented method to synthesize the unique, oxidant-sensitive 6-hydroxy-L-tryptathionine linkage. First, C-6 borylation of a suitably protected L-tryptophan was performed according to recent literature. Then, fluorocyclization of 6-boronate-L-tryptophan yielded a fluoropyrrolo indoline (Fpi) moiety that was shown to engage in the Savige-Fontana reaction with trifluoroacetic acid to furnish the 6-boronate-tryptathionine crosslink. In this synthesis, a boronate was used as a latent hydroxy group that could be revealed on the fully elaborated toxin following an oxidative deborylation reaction. In Chapter 4, the first total synthesis of α-amanitin is concluded. First, incorporation of 6-boronate-Fpi yielded a 6-hydroxy-tryptathionine crosslink. Then, the synthetic (2S,3R,4R)-dihydroxyisoleucine was introduced to the peptide sequence of α-amanitin. Following a macrolactamization step and a diastereoselective sulfoxidation of the tryptathionine thioether to the corresponding (R)-sulfoxide found in the natural product, the synthetic α-amanitin was afforded. Juxtaposition of the synthetic and authentic α-amanitins and extensive comparison of their physical, chemical and biological properties validated the synthetic analogue. The analogues of trans-hydroxyproline and the method for their incorporation into α-amanitin derivatives are disclosed in Chapter 5. The hydroxyproline residue of α-amanitin has been shown to be critical for the toxicity of this toxin. However, surprisingly, there is little traction in the literature regarding the structure-activity relationships (SAR) of the hydroxyproline space and how it could affect the binding of the toxin to RNA polymerase II. Hence, a series of hydroxyproline analogues, including a photocleavable hydroxyproline derivative, were synthesized and aimed to be incorporated into amanitin via an improved solid-phase strategy.Science, Faculty ofChemistry, Department ofGraduat

Synthesis of a cytotoxic amanitin for biorthogonal conjugation

Alpha-amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near-native toxicity. A pendant alkyne was readily installed to enable copper-catalyzed alkyne-azide cycloaddition (CuAAC) to azido-rhodamine and two azide-bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.</p

Synthesis of the Death-Cap Mushroom Toxin α‑Amanitin

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, <i>Amanita phalloides</i>. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, <i>A. phalloides</i> has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-trypta­thionine sulfoxide bridge, enantio­selective synthesis of (2<i>S</i>,3<i>R</i>,4<i>R</i>)-4,5-dihydroxy-isoleucine, and diastereo­selective sulfoxidation

Synthesis of the Death-Cap Mushroom Toxin α‑Amanitin

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, <i>Amanita phalloides</i>. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, <i>A. phalloides</i> has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-trypta­thionine sulfoxide bridge, enantio­selective synthesis of (2<i>S</i>,3<i>R</i>,4<i>R</i>)-4,5-dihydroxy-isoleucine, and diastereo­selective sulfoxidation