Formulating with Less API: Exploring the Application of API Sparing Techniques in Oral Drug Development

Lisa Caralli - Senior Director, Scientific Advisory, Catalent; Karl Box - CSO Europe, Pion UK.

In the early stages of pharmaceutical product development, API is typically in scarce supply as synthesis routes are not yet optimized for large-scale production. With only small amounts of valuable API to experiment with, formulators must make every milligram work hard to yield the maximum informational output. Gathering sufficient meaningful data to support effective decision-making around which candidates to take forward into in vivo studies is a major challenge. In silico modeling and representative in vitro testing, both have an important role to play.

In this article we discuss API-sparing in vitro techniques that can effectively support oral drug development, focusing on those associated with optimizing solubility. Of the three factors that define oral bioavailability – solubility, permeability, and metabolism – low aqueous solubility is a concern for approximately 75%¹ of the new molecules coming out of discovery. Since formulators are most able to impact solubility, API-sparing methods are particularly valuable for solubility enhancement studies when developing enabled formulations. We examine the technological advances that are enabling faster, smaller volume testing and present their value through the applications of these techniques.

Making Progress When API is Limited

The pathway of oral drug development, from molecular discovery through pharmacokinetic (PK) and toxicology studies to first-in-human studies, is demanding and time-pressured. Several prototype formulations are typically taken into early PK studies with only limited formulation and in vitro data to guide selection. Subsequent formulation optimization typically proceeds via an iterative process of adjustment and further PK studies. The need to gather relevant data to refine the field of formulation candidates for in vivo studies as early and reliably as possible makes it vital for drug developers to think carefully about how to use any available API to support this process. When API is scarce, what strategies are most helpful in reliably identifying the best candidates, supporting formulation, and maximizing the chances of success of first-in-human studies?

A good starting point is to use in silico modeling to predict Absorption, Distribution, Metabolism, and Excretion (ADME) parameters from the molecular structure of a candidate API. Preliminary models can be established for the intended delivery route, with oral being the preferred route for most drugs. These models provide an initial baseline PK profile that can be refined through the inclusion of in vitro test data. For example, testing with liver microsomes and/or a hepatocyte cell line provides insight into metabolic susceptibility. Solubility data in biorelevant media and utilization of the Caco-2 and other epithelial cell lines to predict intestinal drug permeability provides complementary elucidation of oral absorption potential. Measurements of solubility in, for example, Fasted State Simulated Intestinal Fluid (FaSSIF), Fed State Simulated Intestinal Fluid (FeSSIF), and Simulated Gastric Fluid (SGF) along with permeability data can be incorporated into the Developability Classification System (DCS)² to identify risks to oral bioavailability and guide formulation development work.

Preferred oral drug candidates are typically those that are inherently resistant to hepatic metabolism, are not efflux substrates, and/or display high permeability since these properties are most challenging to alter. In contrast, formulators have multiple tools for solubility enhancement. While API-sparing techniques are a valuable alternative to in vivo permeability assessment, they are particularly helpful for solubility studies.

Technology That Can Make a Difference

The application of API sparing techniques relies on the availability of appropriate technology, specifically small-scale instrumentation capable of delivering accurate, representative data. In recent years significant advances have been made concerning commercially available solutions, so an up-to-date understanding of what is now feasible is useful.

One way to gauge the advancements is by examining the size of the testing apparatus. For instance, the standard USP apparatuses for dissolution testing have a nominal capacity of at least 1L (USP Apparatus 1 and 2) or a flow rate of several hundred mL/h (USP Apparatus 4). These volumes are impractical for extensive studies with small quantities of API since the amount required to achieve detectable concentrations, which can be measured with confidence, is prohibitive. In contrast, state-of-the-art systems, as exemplified by the microDISS Profiler (Pion Inc.) enable reliable dissolution testing with a test volume of just 2 – 20 mL, reducing the amount of API required to around 10 – 100 µg of powder. This is orders of magnitude less material than is required with a conventional test apparatus.

Reducing apparatus volumes to this extent creates a need for corresponding solutions for concentration measurement. Periodic sampling followed by offline analysis – UV or High-Performance Liquid Chromatography (HPLC) – as carried out in standard dissolution testing is challenging, time and labor-intensive, and often suffers from a relatively low time resolution. Rather, fiber optic UV monitoring is increasingly recognized as the preferred alternative. Fiber optic UV monitoring systems measure concentration in real-time in the dissolution media using an in situ dip probe and offer two key advantages. Firstly, they are physically well-suited to small-scale equipment with the probe occupying minimal volume and the best systems simultaneously monitoring multiple individual test vessels for high throughput testing. Secondly, they provide data at a rate that substantially surpasses that achievable with manual sampling. Using a compact small-scale test set-up with UV monitoring, formulation scientists can gather data every few seconds to study dissolution behavior in exceptional detail, even with minimal amounts of API.

Fiber optic UV monitoring systems support in vitro API-sparing permeability assessment in just the same way. The MicroFlux device (Pion Inc.) for example, uses fiber-optic UV probes to simultaneously measure concentration in low-volume donor and receiver chambers separated by a synthetic biomimetic gastrointestinal tract (GIT) permeation membrane. The result is real-time dissolution and flux monitoring, for rapid and effective permeability assessment to support DCS application and inform formulation strategy.

A closer examination of specific test methods and their informational output is helpful in understanding what can be measured and the potential benefits.

A Toolkit of API Sparing Techniques

Using a small volume dissolution apparatus together with an in situ fiber optic monitoring system formulators can rapidly carry out baseline studies of the solubility and dissolution behavior of candidate APIs. Using minimal quantities of API, such tests can efficiently identify promising formulation approaches for solubility enhancement during early phase development such as amorphization to induce transient supersaturation and improve kinetic solubility or micronization to enhance dissolution rate. This test set-up also enables additional experimental strategies and the investigation of various behaviors of direct relevance to oral drug product development.

Two-stage dissolution studies

For ionizable drugs, the investigation of the dissolution behavior under the changing pH conditions of the human gastrointestinal (GI) tract is critical to estimate drug absorption. For example, weakly basic APIs may be highly soluble at the low pH encountered in the stomach but precipitate when exposed to higher pH in the small intestine where most absorption occurs. Two-stage dissolution experiments in simulated GI media elucidate precipitation tendency that can be used as a tool to estimate the in vivo performance of the formulation, differentiating drugs that remain in a supersaturated solution from those that readily precipitate out.

Small-scale dissolution testing is a valuable alternative to traditional USP dissolution testing apparatus when it comes to implementing an API-sparing approach to such studies. This becomes particularly relevant in the development of lipid-based formulations and amorphous solid dispersions. With experimental setups that enable simultaneous testing in multiple vessels, comparative testing of enabled formulations in different media is extremely straightforward. For instance, when screening for the best prototype formulation of a lipid-based formulation, these small-scale methods allow researchers to assess the dissolution behavior of various lipid compositions and their impact on drug solubility. Similarly, in the context of amorphous solid dispersion development, this approach aids in comparing different polymers’ performance in enhancing drug dissolution. Moreover, it is also possible to carry out dynamic studies by dosing with aliquots of buffer that rapidly shift pH to simulate the in vivo environment that the API will encounter. By recreating these pH transitions, researchers can observe how the formulation responds to changing pH conditions and evaluate whether drug release matches the intended release profile.

The overarching goal is to ensure that the API remains supersaturated under the neutral pH conditions representative of those in the small intestine long enough to allow for absorption to occur. In the case where the API is prone to recrystallization, follow-up two-stage dissolution experiments can be a useful tool for formulators to identify excipients or formulation techniques that can help maintain drug supersaturation following a pH shift.

Liquid-liquid phase separation (LLPS) and polymer screening

Liquid-liquid phase separation (LLPS) for a drug occurs when the concentration exceeds the “amorphous solubility” of the compound, resulting in the formation of an API-rich colloidal species within a supersaturated aqueous solution. Rapidly crystallizing drugs will often precipitate to the low solubility crystalline state pbeforereaching the LLPS concentration, substantially reducing the concentration of drug available for absorption. Thus, delaying the onset of recrystallization is critical in our efforts to exploit the supersaturated state and drive absorption when formulating drugs as amorphous systems. Since the amorphous solubility for an API is a function of its chemical environment, investigations of LLPS are influenced by the media used, e.g., FaSSIF or FeSSIF. Testing under different conditions establishes the extent of the crystallization risk and the associated need for formulating with precipitation-inhibiting excipients.

Polymers that prevent recrystallization before LLPs by maintaining API supersaturation in conditions representative of the intestine are promising candidates for incorporation into an amorphous solid dispersion (ASD) to achieve the so-called ‘parachute’ effect (see Figure 1).³ In its crystalline state, a sparingly soluble free-base API may dissolve to some extent in the acidic conditions of the stomach but then quickly recrystallize in the more alkaline pH environment of the intestine. Rendering the drug amorphous may initially improve the apparent solubility by disrupting the crystalline lattice, the so-called ‘spring’ effect, but the resulting supersaturated solution is metastable and at significant risk of recrystallization. Precipitation inhibiting polymers, identified, and screened in an in vitro solvent-shift assay, can be incorporated into formulations to delay the recrystallization event, allowing for increased drug absorption. Confirming this ‘parachute’ effect using two-stage dissolution is key before moving into in vivo studies.

Via small-scale dissolution testing, it is possible to identify APIs that exhibit the ‘spring effect’ and complementary polymers that may be suitable for maintaining supersaturation in the intestine. Scoping drug loading for an ASD via experimentation at various API/polymer ratios is also feasible. Together these techniques can enable substantial progress towards an optimized formulation using minimal amounts of API.

Film casting

Film casting is a further API-sparing technique that complements the use of the aforementioned dissolution testing strategies in ASD development. It involves dissolving the candidate API and polymer in a solvent and dosing an aliquot of the resulting solution onto the surface of either a slide or 96-well plate to produce a film (via solvent evaporation) that can be used to assess the physical stability of API/polymer mixtures. Analysis by polarized light microscopy (PLM), differential scanning calorimetry (DSC), and/or powder x-ray diffraction enables the detection of issues such as phase separation or API crystallization to support polymer selection and optimize drug loading. Slides/plates can be stored at elevated temperature and relative humidity e.g., 40°C/75% to explore stability over a wider range of conditions. Generally, drug molecules with low crystallization tendency are better candidates for ASD formulation so this technique can be helpful in either refining API molecule selection or focusing solubility enhancement strategies productively.

Small volume flux measurements

Though there is rightly considerable focus on solubility and solubility enhancement in oral drug formulation, transit through biological membranes within the GIT is also essential for the API to reach the bloodstream. Studies of flux enable the assessment of permeability to elucidate this behavior and just like dissolution testing can be carried out at a small scale when API is scarce. By simultaneously measuring drug concentration on either side of a biomimetic jejunal-like permeation membrane, systems such as MicroFlux assess absorption potential. The resulting measurements can be used to rank order formulation candidates concerning expected in vivo performance and to assess alternative excipients. Such studies are particularly helpful when it comes to the development of complex lipid formulations for which movement across the membrane is especially sensitive to formulation properties.

In Conclusion

Using state-of-the-art technology, formulators with the requisite know-how are now able to apply a raft of API-sparing techniques to advance with confidence, even when API is limited. Via the careful use of such techniques, APIs, excipients, and prototype formulations can be thoroughly characterized to effectively identify the most promising candidates for oral drug development earlier in the development lifecycle. This approach is both time and cost-effective and preserves higher API-consuming in vivo studies for the candidates that truly merit them.

References

1. Water Insoluble Drug Delivery Technology Review, PharmaCircle, Dec 2022

2. Butler J., Dressman J. The developability classification system: application of biopharmaceutics concepts to formulation development. Journal of Pharmaceutical Sciences. (2010) 99:4940.

3. Karimi-Jafari M., Padrela L., Walker, G., Croker D. Creating Cocrystals: A Review of Pharmaceutical Cocrystal Preparation Routes and Applications. Crystal Growth & Design. (2018) 18:6370.

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