Adding a New Dimension to the Amorphous Solid Dispersion Phase Diagram: Studying Dissolution Kinetics of Crystalline Drugs in a Polymer Matrix Using Temperature Dependent XRPD and DSC.

Hot-melt-extrusion (HME) is an enabling technology used for poorly soluble active pharmaceutical ingredients (APIs) to increase the bioavailability by embedding the drug in a water soluble and often amorphous carrier such as a polymer. Knowledge of the most critical factors impacting the dissolution rate of crystalline API in the polymer during manufacturing will provide useful insight for process improvement. In this study, crystalline APIs (Acetaminophen, APAP and Indomethacin, IMC) were analyzed in a polymeric matrix (Copovidone, PVP-VA64) via X-Ray Powder Diffraction (XRPD) and Differential Scanning Calorimetry (DSC) to follow the dissolution process under various conditions in a down-scaled static laboratory system. The combination of in-situ XRPD measurements and a kinetic model based on DSC data proved to be a suitable tool to investigate the dissolution process and can be applied to various APIs and polymers to avoid residual crystallinity and thermal degradation. Thus, the temperature-composition phase diagram in a thermodynamic equilibrium is augmented by the kinetic component as new dimension. The obtained findings set the foundation for investigating the dissolution kinetics and enable the transition from a static to a dynamic system.

Crystallization Risk Assessment of Amorphous Solid Dispersions by Physical Shelf-Life Modeling: A Practical Approach.

Amorphous solid dispersions (ASDs) of a poorly water-soluble active pharmaceutical ingredient (API) in a polymer matrix can enhance the water solubility and therefore generally improve the bioavailability of the API. Although examples of long-term stability are emerging in the literature, many ASD products are kinetically stabilized, and inhibition of crystallization of a drug substance within and beyond shelf life is still a matter of debate, since, in some cases, the formation of crystals may impact bioavailability. In this study, a risk assessment of API crystallization in packaged ASD drug products and a mitigation strategy are outlined. The risk of shelf-life crystallization and the respective mitigation steps are assigned for different drug product development scenarios and the scientific principles of each step are discussed. Ultimately, the physical stability of ASD drug products during shelf-life storage is modeled. The methodology is based on the quantification of crystal growth kinetics by transmission Raman spectroscopy (TRS), modeling the impact of water sorption on the glass-transition temperature of the ASD, and the prediction of moisture uptake by the packaged ASD drug product during storage. This approach is applied to an ASD of fenofibrate that features both fast API crystallization under accelerated storage conditions and long-term stability in a suitable protective packaging under conventional storage conditions.

Manufacturing Amorphous Solid Dispersions with a Tailored Amount of Crystallized API for Biopharmaceutical Testing.

The preparation of an amorphous solid dispersion (ASD) by dissolving a poorly water-soluble active pharmaceutical ingredient (API) in a polymer matrix can improve the bioavailability by orders of magnitude. Crystallization of the API in the ASD, though, is an inherent threat for bioavailability. Commonly, the impact of crystalline API on the drug release of the dosage form is studied with samples containing spiked crystallinity. These spiked samples possess implicit differences compared to native crystalline samples, regarding size and spatial distribution of the crystals as well as their molecular environment. In this study, we demonstrate that it is possible to grow defined amounts of crystalline API in solid dosage forms, which enables us to study the biopharmaceutical impact of actual crystallization. For this purpose, we studied the crystal growth in fenofibrate tablets over time under an elevated moisture using transmission Raman spectroscopy (TRS). As a nondestructive method to assess API crystallinity in ASD formulations, TRS enables the monitoring of crystal growth in individual dosage forms. Once the kinetic trace of the crystal growth for a certain environmental condition is determined, this method can be used to produce samples with defined amounts of crystallized API. To investigate the biopharmaceutical impact of crystallized API, non-QC dissolution methods were used, designed to identify differences between the various amounts of crystalline materials present. The drug release in the samples manufactured in this fashion was compared to that of samples with spiked crystallinity. In this study, we present for the first time a method for targeted crystallization of amorphous tablets to simulate crystallized ASDs. This methodology is a valuable tool to generate model systems for biopharmaceutical studies on the impact of crystallinity on the bioavailability.

Extraordinary Long-Term-Stability in Kinetically Stabilized Amorphous Solid Dispersions of Fenofibrate.

Inhibition of recrystallization of the drug substance in kinetically stabilized amorphous solid dispersions (ASDs) within and beyond shelf life is still a matter of debate. Generally, these ASD systems are considered to be prone to recrystallization, but examples of their long-term stability are emerging in the literature. Since, in some cases, the formation of crystals may impact bioavailability, recrystallization may present a relevant risk for patients as it potentially lowers the effective dose of the formulation. This study shows that such metastable formulations may indeed remain amorphous even after 15 years of storage under ambient conditions. A formulation of fenofibrate stored for 15 years was compared to a freshly prepared batch. A complete physicochemical characterization regarding content, purity, water content and glass transition was conducted. The emphasis of this physicochemical characterization was on crystallinity as a critical quality attribute: polarized light microscopy (PLM) was used as the standard qualitative method and X-ray powder diffraction (XRPD) as the standard quantitative method. An investigation of the crystal growth kinetics by transmission Raman spectroscopy (TRS) was conducted to build a predictive model. The model was applied successfully to predict the observed physical state of the 15-year-old samples. The observations presented here demonstrate that kinetic stabilization alone is able to prevent crystallization in ASDs over prolonged storage periods, suggesting the need for a reassessment of the risk perception for this kind of ASD formulations.

Frozen in Time: Kinetically Stabilized Amorphous Solid Dispersions of Nifedipine Stable after a Quarter Century of Storage.

Kinetically stabilized amorphous solid dispersions are inherently metastable systems. Therefore, such systems are generally considered prone to recrystallization. In some cases, the formation of crystals will impact the bioavailability of the active pharmaceutical ingredient in these formulations. Recrystallization therefore may present a significant risk for patients as it potentially lowers the effective dose of the pharmaceutical formulation. This study indicates that such metastable formulations may indeed remain fully amorphous even after more than two decades of storage under ambient conditions. Different formulations of nifedipine stored for 25 years were compared with freshly prepared samples. A thorough physicochemical characterization including polarized light microscopy, differential scanning calorimetry, X-ray powder diffraction, and transmission Raman spectroscopy was undertaken. This in-depth characterization indicates no signs of recrystallization in the stored samples. The observations presented here prove that long-term stability of amorphous solid dispersions much beyond the typical shelf life for pharmaceutical formulations is indeed possible by kinetic stabilization alone. These findings implicate a reevaluation of the propensity to recrystallize for kinetically stabilized amorphous solid dispersions.

Unraveling the Effect of Immunogenicity on the PK/PD, Efficacy, and Safety of Therapeutic Proteins.

Biologics have emerged as a powerful and diverse class of molecular and cell-based therapies that are capable of replacing enzymes, editing genomes, targeting tumors, and more. As this complex array of tools arises a distinct set of challenges is rarely encountered in the development of small molecule therapies. Biotherapeutics tend to be big, bulky, polar molecules comprised of protein and/or nucleic acids. Compared to their small molecule counterparts, they are fragile, labile, and heterogeneous. Their biodistribution is often limited by hydrophobic barriers which often restrict their administration to either intravenous or subcutaneous entry routes. Additionally, their potential for immunogenicity has proven to be a challenge to developing safe and reliably efficacious drugs. Our discussion will emphasize immunogenicity in the context of therapeutic proteins, a well-known class of biologics. We set out to describe what is known and unknown about the mechanisms underlying the interplay between antigenicity and immune response and their effect on the safety, efficacy, pharmacokinetics, and pharmacodynamics of these therapeutic agents.

Physiologically based pharmacokinetic models of small molecules and therapeutic antibodies: a mini-review on fundamental concepts and applications.

The mechanisms of absorption, distribution, metabolism and elimination of small and large molecule therapeutics differ significantly from one another and can be explored within the framework of a physiologically based pharmacokinetic (PBPK) model. This paper briefly reviews fundamental approaches to PBPK modeling, in which drug kinetics within tissues and organs are explicitly represented using physiologically meaningful parameters. The differences in PBPK models applied to small/large molecule drugs are highlighted, thus elucidating differences in absorption, distribution and elimination properties between these two classes of drugs in a systematic manner. The absorption of small and large molecules differs with respect to their common extravascular routes of delivery (oral versus subcutaneous). The role of the lymphatic system in drug distribution, and the involvement of tissues as sites of elimination (through catabolism and target mediated drug disposition) are unique features of antibody distribution and elimination that differ from small molecules, which are commonly distributed into the tissues but are eliminated primarily by liver metabolism. Fundamental differences exist in the ability to predict human pharmacokinetics based upon preclinical data due to differing mechanisms governing small and large molecule disposition. These differences have influence on the evolving utilization of PBPK modeling in the discovery and development of small and large molecule therapeutics.

Insights into the mechanism of polymer coating self-healing using Raman spectroscopy.

Self-healing polymer coatings are an emerging class of smart materials. Upon mechanical damage the material properties may be restored by self-healing, which can be triggered externally, e.g., by an increased temperature. An alternative approach relies on embedding capsules with repair agents into the polymers, the rupture of which is induced by the mechanical damage, and the release of the repair agents triggers the self-repair reaction. The work at hand presents in situ Raman spectroscopic investigations on the reaction dynamics in such self-healing polymer coatings. Analysis of the Raman spectra allows one to assign specific Raman bands characteristic for the progress of the self-healing reaction.

New challenges and opportunities in nonclinical safety testing of biologics.

New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rd European BioSafe Annual General Membership meeting in November 2013 in Berlin: (i)Approaches to refine use of non-human primates in non-clinical safety testing of biologics and current experience on the use of minipigs as alternative non-rodent species.(ii)Tissue distribution studies as a useful tool to support pharmacokinetic/pharmacodynamic (PKPD) assessment of biologics, in that they provide valuable mechanistic insights at drug levels at the site of action.(iii)Mechanisms of nonspecific toxicity of antibody drug conjugates (ADC) and ways to increase the safety margins.(iv)Although biologics toxicity typically manifests as exaggerated pharmacology there are some reported case studies on unexpected toxicity.(v)Specifics of non-clinical development approaches of noncanonical monoclonal antibodies (mAbs), like bispecifics and nanobodies.

Ru dye functionalized Au-SiO2@TiO2 and Au/Pt-SiO2@TiO2 nanoassemblies for surface-plasmon-induced visible light photocatalysis.

The most commonly used material in photocatalysis is TiO2. Since TiO2 absorbs only UV-light, photosensitizers are used to extend these catalysts' absorption properties into the Vis/NIR spectral range. In this work we merge the commonly used approach of dye sensitization with the only recently developed approach of functionalizing the catalyst with plasmonically active metal nanoparticles in order to achieve synergistic effects between these two types of visible light sensitization. To this end SiO2@TiO2 nanostructures are functionalized with gold nanoparticles or a combination of gold/platinum nanoparticles loaded with Ru dyes and thoroughly characterized by means of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) imaging as well as energy dispersive X-ray spectroscopy (EDX), UV/VIS and surface enhanced resonance Raman scattering (SERRS) spectroscopy. The photocatalytic performance is tested by applying the benchmark experiment of methylene blue degradation. Spectroscopic investigations and electron microscopy proof the successful synthesis of the envisioned structure. The photocatalytic activity of the nanostructures shows up to 52% higher first order rate constants compared to the corresponding nanostructures without further dye functionalization.

Current challenges and opportunities in nonclinical safety testing of biologics.

Nonclinical safety testing of new biotherapeutic entities represents its own challenges and opportunities in drug development. Hot topics in this field have been discussed recently at the 2nd Annual BioSafe European General Membership Meeting. In this feature article, discussions on the challenges surrounding the use of PEGylated therapeutic proteins, selection of cynomolgus monkey as preclinical species, unexpected pharmacokinetics of biologics and the safety implications thereof are summarized. In addition, new developments in immunosafety testing of biologics, the use of transgenic mouse models and PK and safety implications of multispecific targeting approaches are discussed. Overall, the increasing complexity of new biologic modalities and formats warrants tailor-made nonclinical development strategies and experimental testing.

An algorithm for evaluating potential tissue drug distribution in toxicology studies from readily available pharmacokinetic parameters.

Having an understanding of drug tissue accumulation can be informative in the assessment of target organ toxicities; however, obtaining tissue drug levels from toxicology studies by bioanalytical methods is labor-intensive and infrequently performed. Additionally, there are no described methods for predicting tissue drug distribution for the experimental conditions in toxicology studies, which typically include non-steady-state conditions and very high exposures that may saturate several processes. The aim was the development of an algorithm to provide semiquantitative and quantitative estimates of tissue-to-plasma concentration ratios (Kp ) for several tissues from readily available parameters of pharmacokinetics (PK) such as volume of distribution (Vd ) and clearance of each drug, without performing tissue measurement in vivo. The computational approach is specific for the oral route of administration and non-steady-state conditions and was applied for a dataset of 29 Genentech small molecules such as neutral compounds as well as weak and strong organic bases. The maximum success rate in predicting Kp values within 2.5-fold error of observed Kp values was 82% at low doses (<100 mg/kg) in preclinical species. Prediction accuracy was relatively lower with saturation at high doses (≥100 mg/kg); however, an approach to perform low-to-high dose extrapolations of Kp values was presented and applied successfully in most cases. An approach for the interspecies scaling was also applied successfully. Finally, the proposed algorithm was used in a case study and successfully predicted differential tissue distribution of two small-molecule MET kinase inhibitors, which had different toxicity profiles in mice. This newly developed algorithm can be used to predict the partition coefficients Kp for small molecules in toxicology studies, which can be leveraged to optimize the PK drivers of tissue distribution in an attempt to decrease drug tissue level, and improve safety margins.

Confounding parameters in preclinical assessment of blood-brain barrier permeation: an overview with emphasis on species differences and effect of disease states.

Drug delivery across the brain-blood interfaces is a complex process involving physicochemical drug properties, transporters, enzymes, and barrier dysfunction in diseased conditions. Intact blood-brain barrier (BBB) limits the entry of potentially harmful compounds into the brain but may also reduce the CNS permeability of therapeutic agents. BBB permeability is typically assessed by measuring brain-to-plasma ratio in rodents (referred to as B/P ratio, BB, or Kp, often calculated as logBB), an approach that suffers significant limitations as discussed in the present review. Kp is not a permeability measurement but a partition coefficient mainly driven by the relative binding to plasma and brain tissue components including lipids, phospholipids, and proteins. Compounds with high Kp are often lipophilic with low free fraction available to mediate CNS activities. Efforts should be more concentrated on measuring pharmacologically relevant free drug concentrations at the target site. Using healthy rodents to predict brain penetration in patients might be biased due to species differences in BBB-related parameters such as transporter expression and functional activities. In addition, pathophysiological conditions such as aging, multiple sclerosis, and Alzheimer's and Parkinson's diseases have been described to affect BBB permeability, with barrier leakage and altered transporter activity. The impact of these species differences and disease states on drug delivery to the brain is largely overlooked. More data are needed to better understand their clinical implication in order to design more appropriate screening strategies and ultimately better mitigate the risk for failure in late stage development.

Antitumor activity of targeted and cytotoxic agents in murine subcutaneous tumor models correlates with clinical response.

PURPOSE: Immunodeficient mice transplanted with subcutaneous tumors (xenograft or allograft) are widely used as a model of preclinical activity for the discovery and development of anticancer drug candidates. Despite their widespread use, there is a widely held view that these models provide minimal predictive value for discerning clinically active versus inactive agents. To improve the predictive nature of these models, we have carried out a retrospective population pharmacokinetic-pharmacodynamic (PK-PD) analysis of relevant xenograft/allograft efficacy data for eight agents (molecularly targeted and cytotoxic) with known clinical outcome. EXPERIMENTAL DESIGN: PK-PD modeling was carried out to first characterize the relationship between drug concentration and antitumor activity for each agent in dose-ranging xenograft or allograft experiments. Next, simulations of tumor growth inhibition (TGI) in xenografts/allografts at clinically relevant doses and schedules were carried out by replacing the murine pharmacokinetics, which were used to build the PK-PD model with human pharmacokinetics obtained from literature to account for species differences in pharmacokinetics. RESULTS: A significant correlation (r = 0.91, P = 0.0008) was observed between simulated xenograft/allograft TGI driven by human pharmacokinetics and clinical response but not when TGI observed at maximum tolerated doses in mice was correlated with clinical response (r = 0.36, P = 0.34). CONCLUSIONS: On the basis of these analyses, agents that led to greater than 60% TGI in preclinical models, at clinically relevant exposures, are more likely to lead to responses in the clinic. A proposed strategy for the use of murine subcutaneous models for compound selection in anticancer drug discovery is discussed.

Anti-neuropilin-1 (MNRP1685A): unexpected pharmacokinetic differences across species, from preclinical models to humans.

PURPOSE: To compare the pharmacokinetics (PK) of MNRP1685A, a human monoclonal antibody (mAb) against neuropilin-1 (NRP1), in mice, rats, monkeys, and cancer patients from a Phase I study to model with parallel linear and nonlinear clearances. METHODS: Binding characteristics of MNRP1685A in different species were evaluated using surface plasmon resonance technology. PK profiles of MNRP1685A after single and/or multiple doses in different species were analyzed using population analysis. PK parameters were compared across species. RESULTS: MNRP1685A binds to NRP1 in all four species tested. Consistent with the wide expression of NRP1, MNRP1685A demonstrated pronounced non-linear PK over a wide dose range. PK profiles are best described by a two-compartment model with parallel linear and nonlinear clearances. Model-derived PK parameters suggest similar in-vivo target expression levels and binding affinity to target across all species tested. However, compared to typical human/humanized mAbs, non-specific clearance of MNRP1685A was faster in mice, rats, and humans (60.3, 19.4, and 8.5 ml/day/kg), but not in monkeys (3.22 ml/day/kg). CONCLUSIONS: Monkey PK properly predicted the target-mediated clearance of MNRP1685A but underestimated its non-specific clearance in humans. This unique PK property warrants further investigation of underlying mechanisms.

Pharmacokinetics and toxicology of therapeutic proteins: Advances and challenges.

Significant progress has been made in understanding pharmacokinetics (PK), pharmacodynamics (PD), as well as toxicity profiles of therapeutic proteins in animals and humans, which have been in commercial development for more than three decades. However, in the PK arena, many fundamental questions remain to be resolved. Investigative and bioanalytical tools need to be established to improve the translation of PK data from animals to humans, and from in vitro assays to in vivo readouts, which would ultimately lead to a higher success rate in drug development. In toxicology, it is known, in general, what studies are needed to safely develop therapeutic proteins, and what studies do not provide relevant information. One of the major complicating factors in nonclinical and clinical programs for therapeutic proteins is the impact of immunogenicity. In this review, we will highlight the emerging science and technology, as well as the challenges around the pharmacokinetic- and safety-related issues in drug development of mAbs and other therapeutic proteins.

Population pharmacokinetic and pharmacodynamic modeling of MNRP1685A in cynomolgus monkeys using two-target quasi-steady-state (QSS) model.

MNRP1685A (anti-NRP1) is a fully human IgG1 monoclonal antibody against neuropilin-1 (NRP1), a protein necessary for blood vessel maturation. MNRP1685A binds to free membrane-bound NRP1 (mNRP1) and circulating NRP1 (cNRP1). Total cNRP1 increased in a dose-dependent manner following anti-NRP1 administration in mice, rats, and monkeys. The purpose of this study is to develop a mechanism-based model to simultaneously describe the kinetics of both unbound drug (MNRP1685A) and total cNRP1 in cynomolgus monkeys. Pharmacokinetic (PK) and pharmacodynamic (PD) profiles after single- or multiple-dose administrations were well described by the two-target quasi-steady-state (QSS) model. The estimated nonspecific clearance was 3.26 mL/day/kg and central compartment volume was 38.2 mL/kg. The maximum elimination rate for mNRP1-mediated disposition was 98.8 nM/day. The synthesis rate (3.8 nM/day), degradation rate constant (1.53 day(-1)), and complex elimination rate constant (0.260 day(-1)) for cNRP1 were also derived from the model. QSS constants were 6.94 nM for mNRP1 and 2.8 nM for cNRP1. The results suggest that cNRP1 has minimal effect on MNRP1685A PK while mNRP1 plays a major role in the target-mediated drug disposition. This finding is favorable as the desired pharmacological target is mNRP1, rather than cNRP1. The two-target QSS model provides mechanistic understanding of the nonlinear PK of MNRP1685A. Based on the model prediction, the free drug concentrations to maintain free mNRP1 and cNRP1 below 10% of baseline level are 10 and 20 µg/mL, respectively. This serves as a target concentration for clinical dose selection, assuming cynomolgus monkeys are predictive for humans.

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration.

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

Subcutaneous bioavailability of therapeutic antibodies as a function of FcRn binding affinity in mice.

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid ß monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.