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Detrimental Effects of Elevated Temperatures on the Structure and Activity of Phenylalanine Ammonia Lyase-Bovine Serum Albumin Mixtures and the Stabilizing Potential of Surfactant and Sugars

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Abstract

We propose encapsulating phenylalanine ammonia lyase (PAL)-bovine serum albumin (BSA) mixtures as potential oral therapy for the management of phenylketonuria. PAL will metabolize phenylalanine in the gastrointestinal tract while BSA will minimize product inhibition and allow PAL to work at its Vmax. We intend manufacturing microcapsules using spray drying and the proteins will be exposed to heat. In the current pre-formulation studies, we determined the effect of elevated temperatures on the structure and activity of PAL-BSA mixtures and evaluated the stabilizing potential of excipients. Exposure of PAL to 75°C decreased its Vmax. BSA exacerbated the elevated temperature-mediated decrease in PAL Vmax and completely lost the ability to protect PAL from trans cinnamic acid (TCA)-mediated product inhibition. Circular dichroism studies revealed that elevated temperatures did not affect the secondary structure of PAL but decreased BSA α-helicity. Binding experiments showed that elevated temperature-mediated loss in BSA α-helicity was associated with markedly decreased binding and sequestration of TCA, which accounts for the inability of BSA to relieve PAL product inhibition. Sucrose, trehalose, and low concentrations of sodium dodecyl sulfate conferred concentration dependent stabilization of BSA secondary structure against thermal denaturation. The sugars enhanced PAL Vmax, markedly improved TCA binding to BSA, and restored the ability of BSA to relieve PAL product inhibition. PAL-BSA mixtures exposed to elevated temperatures in the presence of sucrose and trehalose exhibited high and constant PAL activity. The results justify inclusion of these sugars in the eventual microcapsule manufacturing process.

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Abbreviations

BSA:

Bovine serum albumin

BH4:

Tetrahydrobiopterin

CD:

Circular dichroism

g.i.t.:

Gastrointestinal tract

Km:

Michaelis constant

PAH:

Phenylalanine hydroxylase

Phe:

Phenylalanine

PAL:

Phenylalanine ammonia lyase

PKU:

Phenylketonuria

s/o:

Solid in oil

sc:

Subcutaneous

SDS:

Sodium dodecyl sulfate

TCA:

Trans cinnamic acid

V max :

Maximum velocity

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Acknowledgements

The authors thank Kyle Yetsko for the analyses and plot of the PAL Circular Dichroism data and for helpful discussions.

Funding

This work was supported by funds from Saint Joseph’s University and from the Department of Pharmaceutical Sciences.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Saint Joseph’s University, University City Campus, Philadelphia, Pennsylvania, 19104, USA

    Amerh Alahmadi & Anil Dmello

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  1. Amerh Alahmadi
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Contributions

Amerh Alahmadi: performed the experiments, plotted figures, conducted statistical analyses of data, and helped with writing the manuscript.

Anil Dmello: conceptualized studies, supervised experiments, and wrote the manuscript.

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Correspondence to Anil Dmello.

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Competing Interests

Research in our laboratory has received the following patent:

U.S. Patent Application No: 15/495,410, allowed October 16, 2018.

Title: Compositions and methods for the treatment of Phenylketonuria (PKU).

The University of the Sciences has licensed the technology to Abri LLC and the corresponding author owns minority stake in the company.

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Alahmadi, A., Dmello, A. Detrimental Effects of Elevated Temperatures on the Structure and Activity of Phenylalanine Ammonia Lyase-Bovine Serum Albumin Mixtures and the Stabilizing Potential of Surfactant and Sugars. AAPS PharmSciTech 23, 297 (2022). https://doi.org/10.1208/s12249-022-02446-y

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