Under the new policy situation, my country’s innovative drugs have sprung up after the rain, but when the preparation process of innovative drugs should be determined and what kind of preparations should be made have always been a big problem for Chinese preparation personnel. In the initial stage of new drug development, the probability of a drug being approved for marketing is still very low. It is not meaningful to spend a lot of time to mature the preparation process, and time does not allow it. Regulations require that the preparation process must be determined before the start of Phase III clinical trials, so what kind of prescription process should we use in the safety evaluation phase, Phase I and Phase II clinical phases to achieve the effect of saving time and trouble? With this question in mind, the author consulted an article titled Discovering and Developing Molecules with Optimal Drug-Like Properties, Chapter 2 Discovery Formulations: Approaches and Practices in Early Preclinical Development. It is now translated into Chinese and recommended to everyone.
2.2 Preclinical formulation strategy
In preclinical trials, the administration of small molecules includes suspensions, solutions, solids or amorphous dispersions, which will be briefly discussed below. There have been a large number of reviews related to formulation development, especially formulation methods related to solubilization. The focus of this chapter is on the application of these methods in non-clinical trials.
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Medicilon Medicilon assists HitGen's HG381 to be approved as China's first clinical STING agonistfor Inhalation Administration Completed the 3Q Verification
Researchers should carefully consider formulating an overall prescription development strategy to support in vivo research in order to reduce time and resources. This strategy should be comprehensive, including early studies such as drug target identification and verification, and long-term toxicity. These studies can ultimately support human clinical trials. In the next part, we will focus on the development of these strategies. After that, we will discuss in detail the actual situation and examples of different types of research, including pharmacology, ADME, toxicology, and alternative drug delivery methods.
2.2.1 Preparation of suspension and nanosuspension
Suspension prescription is the most widely used in the drug discovery stage because it is suitable for various chemical platforms and is easy to prepare. If the solid form of the compound is consistent with the final development, the suspension can replace the standard human administration dosage form (capsule or tablet) to predict exposure. The standard suspension carrier is usually 1-10% cellulose mixture, these celluloses include methyl cellulose, hydroxyethyl cellulose or gum arabic, and a low concentration (0.1%~0.2%w/v) Non-ionic surfactants such as Tween 80 are used as wetting agents. Surfactants can reduce the agglomeration of ionized components due to charges.
Generally, ultrasound can reduce the particle size of the suspension, which is more conducive to the dissolution and absorption of API. The diameter of the particles obtained after ultrasound is about 10 μm. For suspensions, one of the challenges is that the physical properties of early APIs are not ideal, including amorphous or a mixture of amorphous and crystalline forms. If the API is not characterized, then the batch-to-batch variation of the API will make the results of in vivo studies confusing. In addition, the physical stability of the suspension must also be tested to ensure that there is no crystalline transformation, which may affect the exposure. It may not be necessary to determine the properties of these APIs when it is ready for use.
In the drug discovery stage, there is an increasing tendency to use nanoparticle suspensions (Robinow BE, 2004, Nat. Rev. Drug Discov 3(4):353-359). Nanoparticle suspensions are sub-micron colloidal systems. Different from drug micronization, the particle size range of micronization is generally 2~5 μm. Nanoparticles have a larger surface area than larger particles of the same mass, which makes their dissolution rate faster. When the particle size is very small (<200nm), the saturation solubility may increase. According to the Freundlich-Ostwald equation, when the particle size is 100 nm, the saturation solubility increases by approximately 10-15%. (Kesisoglou et al., 2007, Adv. Drug Rev. 59(7):631-644).
Nanoparticle suspensions can be prepared on a small scale in a relatively short period of time using various technologies (Merisko-Liversidege EM et al., 2008, Toxicol Pathol 36(1): 43-48), or they can be given in a variety of ways. Medicine (oral, nasal, intraperitoneal or intravenous injection). The key issue in preparing nanoparticle suspensions is to select (1) the wetting agent for the suspension formulation and (2) the polymer that encapsulates the particles. The polymer should form enough steric hindrance to prevent particle aggregation and particle size growth. The nano-emulsification process should be optimized so that the particle distribution range of the finished product is very narrow. This can minimize Ostwald ripening and particle size growth (Van Eerdenbrugh B et al. 2008, Int. J Pharm 364(1):64-75). In vitro screening should be performed under appropriate biologically relevant conditions and the prescription should be characterized to ensure that particles will not aggregate during the administration process (Kesisoglou F et al. 2007, Mol Cancer Ther 6(7):2012-2021 ). The physical stability of the prescription should be monitored to ensure that during the shelf life, there will be no crystal form transformation or particle size growth. The preparation of nanosuspensions used in the drug discovery phase will be discussed in Chapter 3.
2.2.2 Choose the appropriate pH
Compounds with pKa in the biologically relevant range can usually be prepared into solutions by adjusting pH. The Henderson-Hasselbach equation describes the pH, pKa, and the ratio of ionic and non-ionic compounds in solution. The solubility of ionic compounds is usually greater than that of molecular compounds. The pH is usually adjusted to increase the concentration of ionic species to achieve higher solubility. Dilute sodium hydroxide or hydrochloric acid solutions are often used to adjust pH. The pH of the finished product should be monitored to ensure that it is within the acceptable pH range for test animals. The chemical stability of the compound under this pH condition should be confirmed. For compounds with higher pH-dependent solubility in the pH range of the gastrointestinal tract (2-7), a buffer system should be used in short-term trials to minimize the risk of precipitation in the gastrointestinal tract. For intravenous prescriptions, if the pH is outside of 6-8, the buffer capacity should be low enough (usually the molar concentration should be about 25 mM or lower) so that it will not irritate the veins.
Non-ionic molecules are more easily absorbed through the gastrointestinal mucosa. In order to understand the effect of pH changes on solubility, it is necessary to consider the behavior of molecules of different forms in equilibrium. The pH at which both ionized and non-ionized molecules reach saturation is called pHmax. For basic compounds, when the gastrointestinal tract is switched, it enters a higher pH region from the environment of pHmax, and the equilibrium solubility at the corresponding pH may cause crystallization. For general compounds, there will be a supersaturated metastable state, which will last for a period of time, depending on the intrinsic properties of the compound and the environment. This increases drug flux and exposure (Pole DL 2008, J Pharm Sci 97(3):1071-1088).
However, the supersaturation state can cause the precipitation of acidic drugs in the stomach and the precipitation of basic drugs in the intestines, which may result in low exposure and/or greater variability. Adding polymers to the drug delivery system can often prevent and reduce precipitation.
Another effective way to adjust the pH to obtain a solution is in-situ salt formation. The method is by adding a counter ion acid or base to the free base or free acid compound. For ionic compound solutions, the corresponding counterions are different, and the saturation solubility is also different. This method may be useful if a compound in its free form is difficult to formulate (for example, poor suspendability, high viscosity in the administration medium). It is impractical to perform traditional salt-type screening in the early discovery stage. It can be better solved by in-situ salt formation. Generally, the pKa of the counterion should be lower or higher than the pKa of the free compound by more than 2 units. For basic compounds, the pKa of the counter ion should be at least 2 units lower than the pKa of the free base, and for acidic compounds, the pKa of the counter ion should be at least 2 units higher than the pKa of the free acid. Due to the small amount of materials required, different counter ions can be screened to obtain the best combination. It should be emphasized that the primary purpose of this method is to find the prescription with the best solubility, so the long-term variability of the selected salt type is not considered.
2.2.3 Preparation of cyclodextrin complex
Cyclodextrin is a ring-shaped oligoglucose, which is widely used in drug solubilization (Stella et al. 2008, Toxicol Pathol 36(1): 30-42). Cyclodextrin has a hydrophilic outer edge and a hydrophobic inner core. Therefore, its main solubilization mechanism is the formation of non-covalently bonded inclusion compounds with lipophilic drugs. If the drug and cyclodextrin molecules form a complex at a ratio of 1:1, then the solubility has a linear relationship with the concentration of cyclodextrin. The main advantage of this method is that the risk of drug precipitation during absorption is low. When administered by injection, the main reason for the dissociation of the complex is the combination with plasma during absorption and competition (Stella et al. 1999, Adv DrugDeliv Rev36(1):3-16). In most cases, the dissociation is complete and the drug is released quickly. However, in some cases, the binding constant (K) of the drug and cyclodextrin is very high (>1×105 M-1), and its impact on drug disposal has been reported (Chapman et al. 2006, J Pharm Sci 95( 2):256-257). In a wonderful review, the importance of the binding constant is described in detail, and the author claims in the article that if the binding constant is less than 1×10-4 M-1, then most of the poorly soluble drugs can be administered with cyclodextrin complexes. Improve oral bioavailability (Carrier et al. 2007, J Control Release 123(2):78-99). The concentration of the drug and cyclodextrin in the prescription is equally important because it affects the balance of the complex. If the degree of absorption is very small (such as in the nasal cavity, in the muscle), or if the concentrated cyclodextrin is taken orally, the behavior in the body may be affected. The most commonly used in the drug discovery and development stage are 2-hydroxypropyl-β-cyclodextrin (HPβCD) and sulfonate butyl-β-cyclodextrin. These cyclodextrins have excellent water solubility, with solubility above 500 mg/mL, and their safety has been extensively studied. Both cyclodextrins have been used in intravenous prescriptions marketed in the United States. Preclinical data show that intravenous administration of sulfonate butyl-β-cyclodextrin has a lower probability of in vitro hemolysis compared with 2-hydroxypropyl-β-cyclodextrin (Luck et al. 2010, J Pharm Sci 99(8):3291-3301).
The commonly used cyclodextrin concentration is about 10-20% w/v. For example, if the target concentration of a drug solubilized with cyclodextrin is 10 mg/mL, its molecular weight is 500, and a 1:1 complex is formed, then the concentration of SBEβCD is 5% w/v or the concentration of HPβCD is 2.8% w/v. Cyclodextrin complexes are often used as a supplementary method for pH adjustment and low-level polymers to increase solubility. Due to the low pKa of the sulfonic acid group, SBEβCD usually has a negative charge at physiological pH, and cationic compounds bind to SBEβCD better than neutral. Therefore, if appropriate, solubility screening should be performed on both the neutral and ionic forms of the compound. In the future, computer simulation methods can be used to guide the screening of compounds.
2.2.4 Surfactant
Surfactants are amphiphilic molecules containing hydrophilic and hydrophobic regions. In an aqueous solution, when the concentration is greater than the critical micelle concentration, the surfactant will form aggregates, such as micelles. The hydrophilic region in the micelle faces the water direction, while the hydrophobic region faces the nucleus. If hydrophobicity is the limiting factor for compound dissolution in aqueous solution, or the molecule itself is amphiphilic, then surfactants are very useful additives. Generally, the apparent solubility of the compound is directly proportional to the concentration of the surfactant. Therefore, prescriptions with surfactants have the least risk of precipitation during absorption. Some authors have listed commonly used surfactants and their oral or intravenous concentrations (Stickley et al. 2008, Annu Rep Med Chem 43:419-451). Commonly used surfactants include Tween 80 and Cremophor EL. Generally, the main challenge of surfactants in pre-clinical prescriptions is the large amount required for solubilization, which is related to tissue tolerance.
Certain surfactants have allergic reactions after intravenous injection, such as Cremophor EL and Tween 80 injected into sensitive animals (especially dogs) and humans. It has been reported that the use of surfactants can change the plasma elimination behavior. Many surfactants have inhibitory effects on the intestinal drug transport process (such as P-glycoprotein transport). This phenomenon has been widely confirmed (Li et al. 2012, Mol Pharm 9(5):1100-1108). Therefore, the pharmacokinetics and pharmacokinetic data obtained in the case of using high concentrations of surfactants should be interpreted with great care. It has been reported that the use of polyethylene glycol vitamin E (Vitamin E-TPGS) to improve the bioavailability of poorly soluble drugs has been reported. However, in the long-term test of oral polyethylene glycol, the absorption of vitamin E is reduced due to the partial hydrolysis of polyethylene glycol. Therefore, when designing a prescription, the nature and concentration of the surfactant should be carefully considered.
2.2.5 Solubilization with co-solvent
In the discovery phase, organic co-solvent systems are very useful. Co-solvents can change the polarity of the aqueous system and provide an environment that is more conducive to the dissolution of non-polar solutes. Most co-solvents are characterized by hydrogen bond donors or acceptors that can interact strongly with water, and are miscible with water at any ratio. The carbon-hydrogen bond region can reduce the ability of the aqueous system to expel non-polar molecules. Therefore, the co-solvent system is a highly feasible and powerful means to increase the solubility of non-polar molecules in aqueous solutions. Common co-solvents include N-methyl-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, PEG400, and dimethylacetamide. Solubility has a log-linear relationship with co-solvent concentration.
Therefore, for co-solvent-based prescriptions, if the prescription concentration is close to its maximum solubility, supersaturation will occur during the absorption process of oral or parenteral administration. Precipitation may occur, resulting in low exposure or large variability. For oral administration, a small amount of polymer or surfactant can be added to slow down the precipitation. The toxicity and tolerance of co-solvents are issues that need to be considered. Usually, the use of co-solvents is restricted in high-dose (large doses of co-solvents must be used) or long-term tests (such as toxicity tests). When used in a prescription for intravenous injection, it may cause hemolysis. Reed and Yalkowsky established an in vitro screening method that can evaluate the risk of prescription hemolysis (1985, J Parenter Sci Technol 39(2): 64-69).
In addition, co-solvents (especially PEG400) can interfere with biological analysis methods due to their ion suppression effect. Since peaks are emitted at the same time as the excipients, the analysis signal response of the target compound will be reduced. If this problem is confirmed, it can be solved by changing the HPLC method and reducing or removing interfering excipients.
2.2.6 Liposome
For molecules with high logP (>4), lipid-based formulations are very attractive. Such prescriptions include simple oil-formed emulsions, microemulsions, self-emulsifying and self-microemulsifying drug delivery systems (SEDDS/SMEDDS). SEDDS and SMEDDS are mixtures of lipids, surfactants and co-solvents. They are dispersed in an aqueous solution to form an emulsion or microemulsion, which can effectively increase the exposure of highly lipophilic molecules. Williams et al. 2013, Pharmacol Rev 65(1) :315-499).
The in vivo behavior of this type of preparation depends on its behavior in the gastrointestinal tract. For example, a formulation composed of long-chain triglycerides can be lipolyzed, and the digested product will be further solubilized by the cholate-lecithin complex, which will form a good colloidal dispersion, which will pass the first pass metabolism , Mainly absorbed through the intestinal lymph. Therefore, the bioavailability of liposomes is greater than that of solubilizing compounds that are absorbed through the standard mechanism of the portal venous system. For example, liposomes of testosterone derivatives are prescribed for oral administration, the target is the lymphatic system, and at the same time, it can reduce the exposure in the liver during first-pass metabolism.
The solubility of the compound suitable for the formulation in the liposome system should be high enough to meet the administration requirements in animal experiments. The physical and chemical stability of the compound in the carrier/administration formulation is sometimes the main obstacle to long-term use and should be carefully studied. Liposomes may reduce the amount of drug used and the duration of the trial, and its clinical pharmacological parameters, safety and tolerability should be evaluated. Despite these obstacles, the advantages of liposomes are great. Chen et al. provided a strategy for including liposomes in the drug discovery flow chart. The nature of certain biological targets determines that they cannot be combined with drugs prepared by ordinary methods. This strategy is extremely valuable (Chen XQ et. al., 2012, J Med Chem 55(18):7945-7956).
2.2.7 Solid dispersion
Oral prescription preparations made from amorphous solid dispersions have attracted great attention in the drug discovery stage. It increases exposure in animal and human trials. Using relatively safe auxiliary materials, solid dispersions can be prepared on a small scale. Amorphous solid dispersion prescription is to disperse the amorphous drug on the carrier matrix. After administration, the API forms a supersaturated solution, increasing the flux of the drug through the intestinal membrane. If the appropriate polymer materials are used, the supersaturated state can be maintained for several hours, thereby overcoming the absorption difficulties caused by low solubility (Vo et al. Eur J Pharm Biopharm 85(3):799-813).
The polymers and other excipients used to prepare amorphous solid dispersions are usually larger than the acceptable daily intake (ADI) for preparing simple prescription excipients described earlier. Solid dispersions are an attractive choice for longer-term experiments. In recent years, many creative products have been put on the market in the form of solid dispersions. The use of solid dispersion formulations in the drug discovery phase has been reported. Our experience at Lilly has shown that amorphous dispersions have been successfully used in oral toxicity and clinical studies, which can significantly improve plasma exposure and reduce variability.
Compared with the formulation methods described above, solid dispersions are more complex. The physical and chemical stability of the solid dispersion formulation should be carefully evaluated to ensure the stability of handling and storage during the research process. Usually, its development process first includes small-scale experiments, selecting a combination of polymers and drugs to achieve the best dissolution profile, and then a larger scale but still milligram-scale experiments to evaluate the thermodynamic properties of the prescription And physical/chemical stability (Qian et al. J Pharm Sci 99(7):2941-2947). To prepare an amorphous solid dispersion, raw and auxiliary materials should be added in excess to make up for the loss during the preparation and processing. Therefore, the choice of this kind of prescription is just that some compounds have no other preparation methods to choose from, or the special needs in in vivo research make the excipients used in other optional preparations unusable.