Recombinant Spider Silk Hydrogels for Sustained Release of Biologicals.

Therapeutic biologics (i.e., proteins) have been widely recognized for the treatment, prevention, and cure of a variety of human diseases and syndromes. However, design of novel protein-delivery systems to achieve a nontoxic, constant, and efficient delivery with minimal doses of therapeutic biologics is still challenging. Here, recombinant spider silk-based materials are employed as a delivery system for the administration of therapeutic biologicals. Hydrogels made of the recombinant spider silk protein eADF4(C16) were used to encapsulate the model biologicals BSA, HRP, and LYS by direct loading or through diffusion, and their release was studied. Release of model biologicals from eADF4(C16) hydrogels is in part dependent on the electrostatic interaction between the biological and the recombinant spider silk protein variant used. In addition, tailoring the pore sizes of eADF4(C16) hydrogels strongly influenced the release kinetics. In a second approach, a particles-in-hydrogel system was used, showing a prolonged release in comparison with that of plain hydrogels (from days to week). The particle-enforced spider silk hydrogels are injectable and can be 3D printed. These initial studies indicate the potential of recombinant spider silk proteins to design novel injectable hydrogels that are suitable for delivering therapeutic biologics.

An in vitro digestion test that reflects rat intestinal conditions to probe the importance of formulation digestion vs first pass metabolism in Danazol bioavailability from lipid based formulations.

The impact of gastrointestinal (GI) processing and first pass metabolism on danazol oral bioavailability (BA) was evaluated after administration of self-emulsifying drug delivery systems (SEDDS) in the rat. Danazol absolute BA was determined following oral and intraduodenal (ID) administration of LFCS class IIIA medium chain (MC) formulations at high (SEDDSH-III) and low (SEDDSL-III) drug loading and a lipid free LFCS class IV formulation (SEDDS-IV). Experiments were conducted in the presence and absence of ABT (1-aminobenzotriazole) to evaluate the effect of first pass metabolism. A series of modified in vitro lipolysis tests were developed to better understand the in vivo processing of SEDDS in the rat. Danazol BA was low (<13%) following oral and ID administration of either SEDDS. Increasing drug loading, ID rather than oral administration, and administration of SEDDS-IV rather than SEDDS-III led to higher oral BA. After pretreatment with ABT, however, danazol oral BA significantly increased (e.g., 60% compared to 2% after administration of SEDDSL-III), no effect was observed on increasing drug loading, and differences between SEDDS-III and -IV were minimal. In vitro digestion models based on the lower enzyme activity and lower dilution conditions expected in the rat resulted in significantly reduced danazol precipitation from SEDDS-III or SEDDS-IV on initiation of digestion. At the doses administered here (4-8 mg/kg), the primary limitation to danazol oral BA in the rat was first pass metabolism, and the fraction absorbed was >45% after oral administration of SEDDS-III or SEDDS-IV. In contrast, previous studies in dogs suggest that danazol BA is less dependent on first pass metabolism and more sensitive to changes in formulation processing. In vitro digestion models based on likely rat GI conditions suggest less drug precipitation on formulation digestion when compared to equivalent dog models, consistent with the increases in in vivo exposure (fraction absorbed) seen here in ABT-pretreated rats.

Non-linear increases in danazol exposure with dose in older vs. younger beagle dogs: the potential role of differences in bile salt concentration, thermodynamic activity, and formulation digestion.

PURPOSE: To explore the possibility that age-related changes in physiology may result in differences in drug bioavailability after oral administration of lipid based formulations of danazol. METHODS: Danazol absorption from lipid formulations with increasing drug load was examined in younger (9 months) and older (8 years) beagles. Age related changes to hepatic function were assessed via changes to systemic clearance and serum bile acid concentrations. Changes to lipolytic enzyme activity and intestinal bile salt concentration were evaluated using in vitro lipolysis. RESULTS: Drug exposure increased linearly with dose in younger animals. In older animals, bioavailability increased with increasing dose to a tipping point, beyond which bioavailability reduced (consistent with initiation of precipitation). No differences in hepatic function were apparent across cohorts. Changes to enzyme concentrations in lipolysis studies had little impact on drug precipitation/solubilisation. In contrast, higher bile salt concentrations better supported supersaturation at higher drug loads. CONCLUSIONS: Differences in animal cohort can have a significant impact on drug absorption from lipid based formulation. For danazol, bioavailability was enhanced under some circumstances in older animals. In vitro experiments suggest that this was unlikely to reflect changes to metabolism or lipolysis, but might be explained by increases in luminal bile salt/phospholipid concentrations in older animals.

Lipid-based formulations and drug supersaturation: harnessing the unique benefits of the lipid digestion/absorption pathway.

Drugs with low aqueous solubility commonly show low and erratic absorption after oral administration. Myriad approaches have therefore been developed to promote drug solubilization in the gastrointestinal (GI) fluids. Here, we offer insight into the unique manner by which lipid-based formulations (LBFs) may enhance the absorption of poorly water-soluble drugs via co-stimulation of solubilization and supersaturation. Supersaturation provides an opportunity to generate drug concentrations in the GI tract that are in excess of the equilibrium crystalline solubility and therefore higher than that achievable with traditional formulations. Incorporation of LBF into lipid digestion and absorption pathways provides multiple drivers of supersaturation generation and the potential to enhance thermodynamic activity and absorption. These drivers include 1) formulation dispersion, 2) lipid digestion, 3) interaction with bile and 4) lipid absorption. However, high supersaturation ratios may also stimulate drug precipitation and reduce exposure where re-dissolution limits absorption. The most effective formulations are likely to be those that generate moderate supersaturation and do so close to the site of absorption. LBFs are particularly well suited to these criteria since solubilization protects against high supersaturation ratios, and supersaturation initiation typically occurs in the small intestine, at the absorptive membrane.

Toward the establishment of standardized in vitro tests for lipid-based formulations. 2. The effect of bile salt concentration and drug loading on the performance of type I, II, IIIA, IIIB, and IV formulations during in vitro digestion.

The LFCS Consortium was established to develop standardized in vitro tests for lipid-based formulations (LBFs) and to examine the utility of these tests to probe the fundamental mechanisms that underlie LBF performance. In this publication, the impact of bile salt (sodium taurodeoxycholate, NaTDC) concentration and drug loading on the ability of a range of representative LBFs to generate and sustain drug solubilization and supersaturation during in vitro digestion testing has been explored and a common driver of the potential for drug precipitation identified. Danazol was used as a model poorly water-soluble drug throughout. In general, increasing NaTDC concentrations increased the digestion of the most lipophilic LBFs and promoted lipid (and drug) trafficking from poorly dispersed oil phases to the aqueous colloidal phase (AP(DIGEST)). High NaTDC concentrations showed some capacity to reduce drug precipitation, although, at NaTDC concentrations ≥3 mM, NaTDC effects on either digestion or drug solubilization were modest. In contrast, increasing drug load had a marked impact on drug solubilization. For LBFs containing long-chain lipids, drug precipitation was limited even at drug loads approaching saturation in the formulation and concentrations of solubilized drug in AP(DIGEST) increased with increased drug load. For LBFs containing medium-chain lipids, however, significant precipitation was evident, especially at higher drug loads. Across all formulations a remarkably consistent trend emerged such that the likelihood of precipitation was almost entirely dependent on the maximum supersaturation ratio (SR(M)) attained on initiation of digestion. SR(M) defines the supersaturation "pressure" in the system and is calculated from the maximum attainable concentration in the AP(DIGEST) (assuming zero precipitation), divided by the solubility of the drug in the colloidal phases formed post digestion. For LBFs where phase separation of oil phases did not occur, a threshold value for SR(M) was evident, regardless of formulation composition and drug solubilization reduced markedly above SR(M) > 2.5. The threshold SR(M) may prove to be an effective tool in discriminating between LBFs based on performance.

Incomplete desorption of liquid excipients reduces the in vitro and in vivo performance of self-emulsifying drug delivery systems solidified by adsorption onto an inorganic mesoporous carrier.

The purpose of the current study was to provide a mechanistic basis for in vitro and in vivo performance differences between lipid-based formulations solidified by adsorption onto a high surface area material and their respective liquid (i.e., nonadsorbed) counterparts. Two self-emulsifying formulations (based on either medium-chain or long-chain lipids) of the poorly water-soluble drug danazol were solidified by adsorption onto Neusilin US2. Liquid and adsorbed lipid-based formulations were subjected to in vitro dispersion-digestion tests, and additional in vitro experiments were performed to elucidate the cause of performance differences. The bioavailability of danazol after oral administration to rats was also assessed. The percentage of the dose solubilized in the aqueous phase during in vitro dispersion-digesting was ∼35% lower for the adsorbed formulations when compared to their liquid counterparts. This trend was also reflected in vivo, where the bioavailability of danazol after administration of the adsorbed formulations was ∼50% lower than that obtained after administration of the equivalent liquid formulation. Incomplete desorption of the microemulsion preconcentrate from the carrier on dispersion-digestion was identified as the main contributor to the reduced pharmaceutical performance of the adsorbed formulations. The results of the current study indicate that solidification of lipid-based formulations through adsorption onto a high surface area carrier may limit formulation (and drug) release in vivo and thereby reduce oral bioavailability.

Lipid digestion as a trigger for supersaturation: evaluation of the impact of supersaturation stabilization on the in vitro and in vivo performance of self-emulsifying drug delivery systems.

The generation of supersaturation in the gastrointestinal (GI) tract is an increasingly popular means of promoting oral absorption for poorly water-soluble drugs. The current study examined the impact of changes to the quantities of medium-chain (MC) lipid (Captex 300:Capmul MCM), surfactant (Cremophor EL) and cosolvent (EtOH), and the addition of polymeric precipitation inhibitors (PPI), on supersaturation during the dispersion and digestion of MC self-emulsifying drug delivery systems (SEDDS) containing danazol. The data suggest that digestion acts as a "trigger" for enhanced supersaturation and that solubilization/precipitation behavior is correlated with the degree of supersaturation on dispersion (S(M)DISP) or digestion (S(M)DIGEST). The ability of the formulation to maintain solubilization in vitro decreased as the S(M) of the formulation increased. PPI significantly increased supersaturation stabilization and precipitation was inhibited where S(M)DISP < 3.5 and S(M)DIGEST < 4. In the presence of polymer, some degree of supersaturation was maintained up to S(M)DIGEST ∼ 8. Differentiation in the ability of SEDDS to maintain drug solubilization stems from the ability to stabilize supersaturation and for MC SEDDS, utilization of lower drug loads, higher surfactant levels (balanced against increases in S(M)DISP), lower cosolvent and the addition of PPI enhanced formulation performance. In vivo studies confirmed the ability of PPI to promote drug exposure at moderate drug loads (40% of saturated solubility in the formulation). At higher drug loads (80% saturation) and in lipid-free SEDDS, this effect was lost, suggesting that the ability of PPIs to stabilize supersaturation in vitro may, under some circumstances, overestimate utility in vivo.

Real time evolution of liquid crystalline nanostructure during the digestion of formulation lipids using synchrotron small-angle X-ray scattering.

The role of the digestion of lipids in facilitating absorption of poorly water-soluble compounds, such as vitamins, is not only an important nutritional issue but is increasingly being recognized as an important determinant in the effectiveness of lipid-based drug formulations. It has been known for some time that lipids often form complex liquid crystalline structures during digestion and that this may impact drug solubilization and absorption. However, until recently we have been unable to detect and characterize those structures in real time and have been limited in establishing the interplay between composition, digestion, and nanostructure. Here, we establish the use of an in vitro lipid digestion model used in conjunction with synchrotron small-angle X-ray scattering by first confirming its validity using known, nondigestible liquid crystalline systems, and then extend the model to study the real time evolution of nanostructure during the digestion of common formulation lipids. The formation of liquid crystalline structures from unstructured liquid formulations is discovered, and the kinetics of formation and dependence on composition is investigated.