Pharmaceutical compounds are molecular solids that frequently exhibit polymorphism of crystal form. One high profile case of polymorphism was ritonavir, a peptidomimetic drug used to treat HIV-1 infection and introduced in 1996. In 1998, a lower energy, more stable polymorph (form II) appeared, causing slowed dissolution of the marketed dosage form and compromising the oral bioavailability of the drug. This event forced the removal of the oral capsule formulation from the market. We have carried out high-throughput crystallization experiments to comprehensively explore ritonavir form diversity. A total of five forms were found: both known forms and three previously unknown forms. The novel forms include a metastable polymorph, a hydrate phase, and a formamide solvate. The solvate was converted to form I via the hydrate phase by using a simple washing procedure, providing an unusual route to prepare the form I ''disappearing polymorph'' Crystals of form I prepared by using this method retained the small needle morphology of the solvate and thus offer a potential strategy for particle size and morphology control. C rystalline polymorphism, or the ability of a compound to exist in multiple solid-state structures (1, 2), has significant impact on the physical properties, performance, and safety of an active pharmaceutical ingredient (API) and its formulated product(s). Hence, control of drug substance polymorphism is of major importance in drug discovery and development and is monitored carefully by the regulatory agencies. Thorough understanding of the relationship between the physical form and the physicochemical and͞or functional properties of an API is critical in selecting the most suitable form for development into a drug product. However, standard industry methods of solid form discovery rely on manual processes that are time consuming and often limited in scope because of the small amounts of material available at early stages of development. To overcome these challenges, high-throughput crystallization systems have been developed (3-5) permitting rapid and more comprehensive exploration of solid form diversity with only small amounts (Ͻ1 mg per trial) of API. Such systems also facilitate evaluation of the utility of all possible physical forms of a drug substance, enable rapid selection of the optimal solid form, and, thus, can accelerate the development process while minimizing the risk of downstream form-related manufacturing and performance issues (2).Ritonavir [Norvir, Abbott Laboratories, North Chicago, IL (5S,8S,10S,11S)-10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid 5-thiazolylmethyl ester] is an important AIDS drug (6, 7) that garnered much attention when a previously unknown, thermodynamically more stable polymorph appeared unexpectedly, having serious implications for the marketed product and the patients taking the drug. During development and initial manufacture of ritonavir, only one monoclinic cryst...

Nanotechnology offers novel delivery vehicles for cancer therapeutics. Potential advantages of nanoscale platforms include improved pharmacokinetics, encapsulation of cytotoxic agents, enhanced accumulation of therapeutics in the tumor microenvironment, and improved therapeutic structures and bioactivity. Here, we report the design of a novel amphiphilic molecule that self-assembles into nanostructures for intracellular delivery of cytotoxic peptides. Specifically, a cationic α-helical (KLAKLAK) 2 peptide that is known to induce cancer cell death by membrane disruption was integrated into a peptide amphiphile (PA) that self-assembles into bioactive, cylindrical nanofibers. PAs are composed of a hydrophobic alkyl tail, a β-sheet forming peptide, and a bioactive peptide that is displayed on the surface of the nanofiber after self-assembly. PA nanostructures that included (KLAKLAK) 2 were readily internalized by breast cancer cells, in contrast to the (KLAKLAK) 2 peptide that on its own was not cell permeable. (KLAKLAK) 2 nanostructures, but not the peptides alone, also induced breast cancer cell death by caspase-independent and Bax/Bak-independent mechanisms associated with membrane disruption. Significantly, (KLAKLAK) 2 nanostructures induced cell death more robustly in transformed breast epithelial cells than in untransformed cells, suggesting a degree of tumor selectivity. Our results provide proof-of-principle that self-assembling PAs can be rationally designed to generate nanostructures that can efficiently deliver cytotoxic peptides to cancer cells. Cancer Res; 70(8); 3020-6. ©2010 AACR.

Self-assembling peptide amphiphile (PA) nanofibers were used to encapsulate camptothecin (CPT), a naturally occurring hydrophobic chemotherapy agent, using a solvent evaporation technique. Encapsulation by PA nanofibers was found to improve the aqueous solubility of the CPT molecule by more than 50-fold. PAs self-assembled into nanofibers in the presence of CPT as demonstrated by transmission electron microscopy. Small-angle X-ray scattering results suggest a slight increase in diameter of the nanofiber to accommodate the hydrophobic cargo. In vitro studies using human breast cancer cells show an enhancement in antitumor activity of the CPT when encapsulated by the PA nanofibers. In addition, using a mouse orthotopic model of human breast cancer, treatment with PA nanofiber encapsulated CPT inhibited tumor growth. These results highlight the potential of this model PA system to be adapted for delivery of hydrophobic therapies to treat a variety of diseases including cancer.

Branched peptide amphiphile (PA) molecules bearing biological epitopes were designed and synthesized using orthogonal protecting group chemistry on amine groups at lysine residues. These molecules self-assemble into high-aspect-ratio cylindrical nanofibers, and their branched architecture enhances accessibility of epitopes for protein binding and also allows the presentation of more than one epitope in a single molecule. The RGDS cell adhesion epitope was used as a model bioactive signal on PA molecules for potential biomedical applications. Aggregation of the branched PA molecules into nanofibers was demonstrated by TEM and through shifts in the protonation profiles of peripheral amines. These systems also formed self-supporting gels in the presence of physiological fluids and other biologically relevant macromolecules such as synovial fluid and DNA, an important property for their potential use in medicine. Fluorescence anisotropy measurements on the PAs with tryptophan residues were performed to examine the effect of branching on packing and mobility of the peptides in the self-assembled nanofibers. The mobility of tryptophan residues was observed to be restricted upon packing of PA molecules into nanofibers. However, relative to linear analogues, branched molecules retain more mobility in the supramolecular aggregates.