Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5°C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak shFcRn binding to CH2 as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins.

Double-stranded RNA (dsRNA) has multiple antitumor mechanisms that may be used to control tumor growth. Previously we have shown that treatment of solid tumors with a plasmid that encodes Sindbis viral RNA replicase complex, pSIN-β, significantly inhibited the growth of tumors in mice. In the present study, we evaluated the feasibility of further improving the antitumor activity of the pSIN-β plasmid by incorporating interleukin-2 (IL2) gene into the plasmid. The resultant pSIN-IL2 plasmid was delivered to mouse melanoma cells that overexpress the sigma receptor. Here we report that the pSIN-IL2 plasmid was more effective at controlling the growth of B16 melanoma in mice when complexed with sigma receptor-targeted liposomes than with the untargeted liposomes. Importantly, the pSIN-IL2 plasmid was more effective than pSIN-β plasmid at controlling the growth of B16 melanoma in mice, and B16 tumor-bearing mice that were treated with pSIN-IL2 had an elevated number of activated CD4(+), CD8(+), and natural killer cells, as compared to those treated with pSIN-β. The RNA replicase-based, IL2-expressing plasmid may have applications in melanoma gene therapy.

Intracellular unbound drug concentrations determine affinity to targets in the cell interior. However, due to difficulties in measuring them, they are often overlooked in pharmacology. Here we present a simple experimental technique for the determination of unbound intracellular drug concentrations in cultured cells that is based on parallel measurements of cellular drug binding and steady-state intracellular drug concentrations. Binding in HEK293 cells was highly correlated with binding in liver-derived systems, whereas binding in plasma did not compare well with cellular binding. Compound lipophilicity increased drug binding, while negative charge and aromatic functional groups decreased binding. Intracellular accumulation of unbound drug was consistent with pH-dependent subcellular sequestration, as confirmed by modeling and by inhibition of subcellular pH gradients. The approach developed here can be used to measure intracellular unbound drug concentrations in more complex systems, for example, cell lines with controlled expression of transporters and enzymes or primary cells.

5-Fluorocytosine (FC), an antifungal drug and a cytosine derivative, has a complex solid-state landscape that challenges its development into a drug product. A total of 8 new FC salts, both cytosinium and hemicytosinium, with four strong acids were prepared by controlling acid concentration in the crystallization medium. We further identified that the pharmaceutically acceptable saccharin salt of FC exhibits supe-rior phase stability and, hence, has the potential to address the instability problem of FC associated with hydration.

The presence of polymers within solid dose forms, such as solid dispersions, or liquid or semi-solid formulations, such as lipid based formulations, can promote the maintenance of drug supersaturation after dissolution or dispersion/digestion of the vehicle in the gastrointestinal tract. Transiently stable supersaturation delays precipitation, increases thermodynamic activity and may enhance bioavailability and reduce variability in exposure. In the current study a diverse range of 42 different classes of polymers, with a total of 78 polymers across all classes, grades and molecular weights were examined, to varying degrees, as potential polymeric precipitation inhibitors (PPIs) using a solvent shift method to initiate supersaturation. To provide a deeper understanding of the molecular determinants of polymer utility the data was also analysed, along with a range of physicochemical descriptors of the polymers employed, using principle component analysis (PCA). Polymers were selectively tested for their ability to stabilise supersaturation for 9 poorly water soluble model drugs, representing a range of non-electrolytes, weak acids and weak bases. In general, the cellulose based polymers (and in particular hydroxypropylmethyl cellulose, HPMC, and its derivatives) provided robust precipitation inhibition across most of the drugs tested. Subsequent PCA indicate that there is consistent PPI behaviour of a given polymer for a given drug type, with clear clustering of the performance of polymers with each of the non-electrolytes, weak bases and weak acids. However, there are some exceptions to this, with some specific drug type - polymer interactions also occurring. Polymers containing primary amine functional groups should be avoided as they are prone to enhancing precipitation rates. An inverse relationship was also documented for the number of amide, carboxylic acid and hydroxyl functional groups, therefore for general good PPI performance the number of these contained within the polymer should be minimised. Molecular weight is a poor predictor of performance, having only a minor influence, and in some cases higher molecular weight enhances the precipitation process. The importance of ionic interactions to the ability of a PPI to stablise the supersaturated state was demonstrated by the advantage of choosing a polymer with an opposite charge with respect to the drug. Additionally, when the polymer charge is the same as the supersaturated drug, precipitation is likely to be enhanced. A PCA model based on polymer molecular properties is presented, which has a central oval region where the polymer will general perform well across all three drug types. If the polymer is located outside of this region, then they either show compound specific inhibition or enhance precipitation. Incomplete separation of the PPI performance based on the molecular properties on the polymers indicates that there are some further molecular properties that might improve the correlation.

One hypothesis for persisting HIV-associated neurocognitive disorders (HAND) in effectively treated individuals is the limited permeation of antiretroviral agents (ARV) across the blood-brain barrier (BBB). However, the physicochemical factors limiting the brain entry of a given ARV and the mutual interactions of combined drugs on their brain entry have not been properly characterized. Using transporter kinetic measurements, we show that large lipophilic drugs such as protease inhibitors (PI) have strong binding affinities to drug efflux transporters expressed at the BBB and thus are prevented from entering the brain. However, when combined, the PI with the highest binding affinity (i.e., boosting ritonavir) will occupy a large proportion of the transporter binding sites and thus slow down the efflux rate of the coadministered PI thereby facilitating its brain entry. Furthermore, using thermodynamic measurements and computational modeling, we show that ARV with small cross-sectional areas (AD < 70 Å(2)) and octanol-water distribution coefficients (-1 < log D <5) such as most nucleoside analogues have a high passive influx and cross the BBB despite interactions with drug transporters. These data indicate that HIV therapies combining small diffusing molecules with large lipophilic molecules are better suited for brain entry and should be preferred for HAND. This work highlights the role of PI as modulators of drugs' brain entry.

Sustained drug delivery to mucosal surfaces has the potential to improve the effectiveness of prophylactic and therapeutic treatments for numerous diseases and conditions, including inflammatory bowel disease, sexually transmitted diseases, cystic fibrosis, glaucoma, dry eye and various cancers. Sustained delivery systems such as nanoparticles can be useful for mucosal delivery, but recent work suggests they should penetrate the rapidly cleared mucus barrier that overlies all mucosal epithelia to achieve uniform distribution on epithelial surfaces and enhanced residence time. Thus, it is important to evaluate mucus-penetrating ability of nano-sized delivery systems in preclinical animal studies, and for administration to humans. We describe a simple ex vivo method to visualize and quantify nanoparticle transport in mucus on fresh mucosal tissues. Using this method in murine models, we observed variations in the mucus mesh at various anatomical locations, as well as cyclical variations that may have implications for mucosal delivery.

Cancer is one of the most common causes of death worldwide. Two types of cancer that have high mortality rates are pancreatic and lung cancer. Despite improvements in treatment strategies, resistance to chemotherapy and the presence of metastases are common. Therefore, novel therapies which target and silence genes involved in regulating these processes are required. Short-interfering RNA (siRNA) holds great promise as a therapeutic to silence disease-causing genes. However, siRNA requires a delivery vehicle to enter the cell to allow it to silence its target gene. Herein, we report on the design and synthesis of cationic star polymers as novel delivery vehicles for siRNA to silence genes in pancreatic and lung cancer cells. Dimethylaminoethyl methacrylate (DMAEMA) was polymerized via reversible addition-fragmentation transfer polymerization (RAFT) and then chain extended in the presence of both cross-linkers N,N-bis(acryloyl)cistamine and DMAEMA, yielding biodegradable well-defined star polymers. The star polymers were characterized by transmission electron microscopy, dynamic light scattering, ζ potential, and gel permeation chromatography. Importantly, the star polymers were able to self-assemble with siRNA and form small uniform nanoparticle complexes. Moreover, the ratios of star polymer required to complex siRNA were nontoxic in both pancreatic and lung cancer cells. Treatment with star polymer-siRNA complexes resulted in uptake of siRNA into both cell lines and a significant decrease in target gene mRNA and protein levels. In addition, delivery of clinically relevant amounts of siRNA complexed to the star polymer were able to silence target gene expression by 50% in an in vivo tumor setting. Collectively, these results provide the first evidence of well-defined small cationic star polymers to deliver active siRNA to both pancreatic and lung cancer cells and may be a valuable tool to inhibit key genes involved in promoting chemotherapy drug resistance and metastases.

In the early drug discovery environment, poorly soluble compounds with suboptimal potency are often used in efficacy studies to demonstrate in vivo preclinical proof-of-concept for new drug discovery targets and in preclinical toxicity studies to assess chemical scaffold safety. These compounds present a challenge to formulation scientists who are tasked with improving their oral bioavailability because high systemic concentrations are required. Despite the use of enabling formulations, increases in systemic exposure following oral delivery are often not achieved. We hypothesize that in some cases non-sink intestinal permeation can occur for poorly soluble compounds where their high systemic concentrations can act to inhibit their own oral absorption. Rats were given a 30 mg/kg oral dose of 1,3-dicyclohexyl urea (DCU) alone or concurrently with deuterated DCU (D8-DCU) intravenous infusions at rates of 13, 17, and 22 mg/kg/h. D8-DCU infusions dose dependently inhibited DCU oral absorption up to a maximum of 92%. Physiologically based pharmacokinetic modeling was utilized to understand the complex interaction between high DCU systemic concentrations and its effect on its own oral absorption. We show that high systemic concentrations of DCU act to suppress its own absorption by creating a condition where intestinal permeation occurs under non-sink conditions. More importantly, we identify relevant DCU concentrations that create the concentration gradient driving the intestinal permeation process. A new parameter, the maximum permeation extraction ratio, is proposed and provides a simple means to assess the extent of non-sink permeation.

The efflux transporter P-glycoprotein (Pgp), encoded by the ABCB1 gene, decreases the bioavailability of a wide range of orally administered drugs. Drug permeability studies using the in vitro Caco-2 cell model commonly rely on small molecule modulators to estimate the contribution of Pgp to drug efflux. The use of such modulators may be limited by their interactions with other membrane transporters. RNA interference, a tool allowing for the specific degradation of a target gene's mRNA, has emerged as a technique to study gene expression and function. This manuscript describes the use of chemically modified small interfering RNA (siRNA) for a dose-dependent suppression of ABCB1 in Caco-2 cells and the subsequent drug permeability assay. We transfected Caco-2 cells while in suspension with chemically modified synthetic siRNA-lipid complexes and then seeded the cells on polycarbonate semipermeable supports. Once the monolayer of Caco-2 cells formed tight junctions and expressed brush border enzymes, we determined the dose-dependent suppression of the ABCB1 gene using RT-qPCR. We measured the duration of silencing at the optimal siRNA dose by Western blot for Pgp protein. The utility of this in vitro model was determined by performing bidirectional transport studies using a well-established substrate for Pgp, rhodamine 123. A single 4 h transfection of the Caco-2 cells with ≥100 nM siRNA reduced the expression of ABCB1 mRNA by >85% at day five in culture. The time-course study revealed that the single transfection reduces Pgp protein levels for 9 days in culture. This magnitude of silencing was sufficient to reduce the efflux of rhodamine 123 as measured by the apparent permeability coefficient and intracellular accumulation. In this study, we demonstrate the dose-dependent, targeted degradation of Pgp in Caco-2 cells as a new model for assessing drug efflux from enterocytes. The dose-dependent nature of the Pgp silencing in this study offers significant improvements over other approaches to creating a Caco-2 model with suppressed ABCB1 expression. We envision that this technique, in conjunction with better small molecule inhibitors, will provide a useful tool for future drug permeability studies.