Formulation Development Strategy: Preclinical PK, PD, and TK Considerations

Preclinical research encompasses crucial studies in pharmacokinetics (PK), pharmacodynamics (PD), toxicology (TOX), and other fields. In addition to in vitro studies, in vivo research provides a more comprehensive assessment of the properties of the API (active pharmaceutical ingredients) and drug products. The formulation development strategy for in vivo preclinical animal studies varies from research objectives¹. For instance, when aiming to attain high oral bioavailability, optimized clear-solution formulations can yield maximal drug exposure and bioavailability and reduce inter-individual variability compared to suspensions. When the research aims to develop candidate drugs into marketable products, various formulation types, such as solutions, suspensions, or solid dosage forms, should be employed to assess the impact of solubility and dissolution rates on their in vivo exposure. Formulation development in such cases necessitates more consideration, and the design of prescription processes is generally more intricate². Therefore, customized formulation strategies are essential for preclinical formulation development to meet the requirements of PK, PD, TOX, and other studies.

Formulation development strategies in pharmacokinetic (PK) studies

In the early drug discovery, after candidate compounds are screened through physicochemical properties and in vitro assessments of DMPK, preliminary PK studies are generally conducted in rodent models. These studies aim to assess candidate compounds' absorption, distribution, metabolism, and excretion properties in vivo, facilitating the selection of high-quality compounds for further research and development. A detailed description of a decision tree used to determine whether a drug can progress to the next stage based on early PK studies in rodents is provided in the literature., as illustrated in Figure 1³.

*Recommended oral bioavailability in mice > 30% (may vary based on disease type and drug potency)

Figure 1. A decision tree for drug development based on early pharmacokinetics studies in rodents

During the early stages, the physicochemical properties, such as particle size distribution, crystalline form, and morphology, change throughout the molecular optimization process. These alterations impact the compound's solubility, dissolution rate, in vivo absorption, and bioavailability, resulting in different PK outcomes. According to the data reported by Merck, MK-0869 (Aprepitant) was prepared in powder form with particle sizes of 5.5 µm, 1.8 µm, 0.48 µm, and 0.12 µm as shown in Figure 2⁴. These different-sized powders were dispersed in a suspension and administered to beagle dogs at 2 mg/kg. The results indicate that as the compound's particle size decreases, the dogs' exposure level increases.

Figure 2. Effect of particle size on blood concentration

Figure 3^{5, 6}, illustrates the influence of compound crystalline forms on PK characteristics based on data reported for Chlortetracycline hydrochloride. The left panel shows the solubility results of two different crystalline forms of the same compound in solution at 37 °C, and the right panel shows the PK results of two different crystalline forms after intraduodenal administration. The results indicate that crystalline form b exhibits higher in vitro solubility and higher in vivo exposure than crystalline form a.

Figure 3. Impact of crystalline form on plasma drug concentration

Therefore, for early PK studies, it is advisable to utilize solution formulations to mitigate the impact of compound properties (solubility, crystalline form, particle size, partition coefficients) on in vivo absorption in animals.

It is imperative to concurrently address the stability of the API and drug product under in vivo physiological conditions. Rapid dilution assays can be conducted in vitro using simulated physiological media. For instance, the solution formulation can be rapidly diluted in 1:1, 1:5, or 1:10 ratios with phosphate buffer, simulated gastric fluid, or simulated intestinal fluid, then assessing their precipitation to obtain a more robust evaluation of the dissolution status in vivo and enabling predictions of its in vivo exposure.

If the in vivo exposure of the test article is low, compound optimization must be carried out based on the specific limiting factors, such as permeability and metabolism. Conversely, if the compound exhibits excellent in vivo exposure in solution formulation, suspension formulations should be employed for the following PK studies: assessing the feasibility of developing solid dosage forms, predicting in vivo exposure for toxicity studies, and determining exposure levels under the maximum tolerated dose. If the exposure in the suspension is not as ideal as it is in solution formulation, advanced equipment such as planetary ball mills or high-pressure homogenizers can be utilized to micronize or nano-size the compound, reducing the particle size and modifying the physical properties to enhance in vivo exposure of the compound ⁵.

As illustrated in Figure 4⁷, developing a compound into various formulations—co-solvent surfactant solution, wet-milled suspension, and nanocrystal dispersion and then dose orally in rats yields distinct PK profiles. Compared to 30 mg/kg wet-milled suspension, 20 mg/kg co-solvent surfactant solution administered as a clear solution enhances the in vivo absorption of the compound. Despite a lower dosage than the suspension, the clear solution state exhibits higher in vivo exposure. The wet-milled suspension and nanocrystal dispersion are both dosed at 30 mg/kg, and the nano-sized formulation with smaller particle size shows optimized physical properties.

Figure 4. The effect of different oral formulations on plasma concentration in rat

Furthermore, at this stage, the screening throughput is large while the quantity is low (1–5 mg), and the time to develop the formulation is tight (24–48 h). Developing an optimal formulation for each compound individually is impractical. Therefore, pharmaceutical companies often develop a set of formulations containing 3–4 excipients for PK studies involving similar or different series of compounds. These formulations include various co-solvents (dimethylacetamide, N-methyl-2-pyrrolidone, ethanol, propylene glycol, polyethylene glycol), surfactants (Tween-80, Kolliphor EL, Solutol® HS 15), cyclodextrins (β-cyclodextrin, sulfobutyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin), and aqueous diluents (buffer solutions, saline, glucose), then dosing through different routes (oral, intravenous, subcutaneous, intramuscular, intraperitoneal).

Table 1. Standardized excipients in single-dose pharmacokinetics studies

Formulation development strategies in pharmacodynamics (PD) studies

PD studies generally involve the determination of the minimum effective dose in a given animal model. Additionally, data from DMPK and CMC should be taken into consideration to determine the optimal time point for blood or other matrix collection. That enables obtaining reliable data on the animal in vivo exposure, establishing PK/PD and dose-response relationships, and assessing the toxicity of a test article in critical organs or tissues.

To validate drug activity and the associated mechanisms, the formulations used in PD studies may differ for different disease types, and factors such as experimental design, species selection, dosage requirements, and duration need to be considered⁸.

In addition to solubility, dilution-precipitation effects, stability, and tolerability of the formulation, the development of formulations in PD studies should avoid interfering with or masking the pharmacodynamic properties or efficacy of the test sample due to the presence of certain excipients. For instance, it is recommended to avoid using sugar or lipid excipients in animal models adapted for diabetes and metabolic disorders. Similarly, in animal pain models, using certain surfactants as excipients is not advised³.

Additionally, formulation optimization strategies can aid in enhancing drug absorption and effect in pharmacological studies, thereby pushing the compounds with therapeutic potential but suboptimal in vitro physicochemical properties to later stages of development. Dehghani et al.⁹ used the lipophilic hydrophobic BCS II class drug tamoxifen citrate (TMC) as a model compound, and employed sesame oil, Tween 80, and edible glycerol as the oil phase, surfactant, and co-surfactant, respectively, to prepare a water-in-oil type microemulsion formulation for the treatment of breast cancer in BALB/C mice. Female BALB/C mice with breast cancer were used, and the study explored the impact of an orally administered TMC microemulsion formulation on the efficacy of breast cancer treatment, comparing it with a non-drug-loaded blank microemulsion control and a commercially available TMC tablet. The results presented in Figure 5 indicate the tumor volume in the non-drug-loaded blank microemulsion group significantly increased after five weeks, and the body weight decreased to some extent, suggesting that the blank microemulsion carrier had almost no efficacy. The orally administered TMC tablet showed a certain degree of inhibition of tumor growth, but the body weight of the mice decreased. The commercially available TMC tablet exhibited some efficacy. In comparison, the TMC microemulsion formulation significantly inhibited tumor growth and avoided the decrease in mouse body weight to some extent, demonstrating superior efficacy. These results suggest that formulation optimization can enhance drug efficacy by reducing particle size and increasing solubility.

Figure 5. Tumor volumes and mouse body weights after 2 and 5 weeks in 3 groups after administration of different preparations of tamoxifen citrate (TMC)

Formulation development strategies in toxicology (TOX) studies

Prior to applying for an investigational new drug for clinical studies, regulatory agencies require TOX studies in rodents and non-rodents to evaluate the potential safety of the test article and the no observed adverse effect level (NOAEL), a phase that requires that the animal be given a dose high enough to achieve a high level of systemic exposure. TOX studies during formulation development need to take full account of the characteristics of larger administration volumes and longer administration times to avoid abnormal animal reactions caused by factors other than the test sample itself.

Simple and safe solutions or suspensions are generally preferred for formulations used in acute and long-term TOX studies. However, compounds with poor solubility, poor permeability, or low systemic exposure require formulation measures to increase their in vivo exposure. The strategy of solubilization enhancement for poorly soluble compounds is similar to the formulation development used in PK studies and involves selecting suitable solvents, reducing particle size, or developing new dosage forms. For poorly absorbed, strongly metabolized compounds with low exposure to systemic circulation, in vivo exposure can be increased by developing the compounds into formulations such as self-emulsifying microemulsions, lipid-based formulation systems, encapsulations, solid dispersions, or nanoparticle delivery systems.

Figure 6 shows publicly disclosed data from Merck¹⁰, where a difficult-to-dissolve drug (solubility in all vehicles is less than 1 mg/mL) was formulated and optimized to examine its in vivo exposure in male Wistar rats. The data show that self-emulsifying drug delivery systems or the development of nanoparticle delivery systems can significantly increase the level of systemic exposure at low (10 mg/kg) and high (100 mg/kg) doses, compared with the use of a surfactant or co-solvent solubilization to develop solutions. In particular, the development of nanoparticle delivery systems can significantly increase systemic exposure at a dose of 100 mg/kg, effectively meeting the needs of TOX experiments that require higher systemic exposure levels.

Figure 6. Oral exposure in rats for an insoluble compound (solubility <1 mg/mL in all vehicles) using various enabled formulations [male Wistar rats, 10 and 100 mg/Kg

(MPK)].

Abbreviations: AUC, area under the curve; SMEDDS, self-emulsifying drug delivery system.

In practical applications, it is necessary to develop suitable formulations for TOX studies based on the nature of the compounds, the choice of species, and the specific experimental design.

Summary

The DMPK Service Department of WuXi AppTec has established a solvent formulation screening and PK evaluation system. Based on extensive project experience, the team has created a database of combination solvents for preclinical animal experiments. This database allows the selection of animal-tolerable solvents suitable for preclinical PK studies within one working day. Additionally, it can screen more than ten different combinations of solvents for preliminary toxicity studies within 3–5 working days.

The formulation development service of WuXi STA possesses expertise in drug solid-state chemistry, physicochemical characterization, and various formulation development technologies. This includes conventional tablets, capsules, modified-release coatings, spray-dried solid dispersions, hot-melt extrusion technology, nano-suspensions, liquid-filled capsules, pediatric formulations, and high-potency sterile injectables, offering a range of formulation development and manufacturing services. The IND Enabling Preformulation Package (IEPP) platform of WuXi STA provides an end-to-end compound-to-drug evaluation process for new chemical entities (NCE). The IEPP platform can help partners identify challenges and risks in the drug development process as early as possible, and seek suitable solutions in time to advance the process of bringing new drugs to market at a more optimized cost.

In the process of formulation preparation and optimization, WuXi AppTec DMPK and WuXi STA work closely together, integrating technologies and platforms and optimizing time and cost through cross-departmental resource sharing to accelerate drug development and filing by facilitating the entire process from solvent and formulation screening to clinical filing.

Talk to a WuXi AppTec expert today to get the support you need to achieve your drug development goals.

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Authors: Juanjuan Zhang, Binbin Tian, Guang Yang, Jing Jin, Tianjing Zhao, Liang Mao

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,500+ global clients, and have successfully supported 1,200+ IND applications.