DXd, a camptothecin derivative, is employed as the payload molecule for the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd), which is in current clinical use for the treatment of breast and gastric cancer. D...DXd, a camptothecin derivative, is employed as the payload molecule for the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd), which is in current clinical use for the treatment of breast and gastric cancer. Despite the clinical success of ADCs for treatment of cancer, exposure to released payload in plasma and extracellular fluids contributes to off-target toxicities, which limit ADC dosage and efficacy. In this study, we evaluated an anti-DXd antibody fragment, 8C2 Fab, for utility in mitigating DXd toxicity in cell culture and for improving the therapeutic index of T-DXd in vivo in mice. 8C2 Fab was produced, purified, and characterized for binding to DXd and T-DXd. In vitro competitive cytotoxicity assays showed that 8C2 Fab blocked the cell-killing effect of DXd, increasing the DXd concentration associated with 50% growth inhibition (IC) by 50-fold; however, 8C2 did not decrease the cytotoxicity of T-DXd. Co-administration of 8C2 Fab to mice with 600 mg/kg T-DXd significantly decreased the percentage body weight loss at nadir in mice compared to results found with T-DXd alone (12.8 ± 3.66% vs 7.08 ± 4.24%, p = 0.0331). In contrast, co-administration of 8C2 Fab with T-DXd (1 or 10 mg/kg) did not negatively impact the anti-tumor efficacy in mice bearing NCI-N87 xenograft tumors. Our results demonstrate that 8C2 Fab decreases DXd cytotoxicity in vitro, and decreases T-DXd-induced toxicity in vivo, while not inhibiting T-DXd anti-tumor efficacy in vitro or in vivo, supporting utility for improving the therapeutic index of T-DXd.
A bottom-up physiologically based pharmacokinetic (PBPK) model was developed to predict the pharmacokinetics of loperamide, a substrate for multidrug resistance 1 (Mdr1) encoded P-glycoprotein (P-gp), in wild-type dogs (...A bottom-up physiologically based pharmacokinetic (PBPK) model was developed to predict the pharmacokinetics of loperamide, a substrate for multidrug resistance 1 (Mdr1) encoded P-glycoprotein (P-gp), in wild-type dogs (WT) and dogs that are homozygous for a base-pair deletion in the Mdr1 gene encoding for P-gp (Mu, Δ-Mdr1). In vitro-to-in vivo extrapolation (IVIVE) techniques were employed where in vitro data describing loperamide absorption, distribution, metabolism, and elimination (ADME) were extrapolated to in vivo dose exposure predictions. Importantly, by applying system parameters extrapolated from other breeds and published information on Collie-specific physiology, for the first time, a breed-specific whole-body PBPK model for the Collie was developed. Using our loperamide IVIVE-PBPK model (Simcyp Animal Simulator), the observed plasma concentration-versus-time profiles after intravenous and oral loperamide administration were successfully captured. The overall model performance for the WT (n = 7) and Mu (n = 10) Collies was within 1.40 and 1.24, and 1.18 and 1.51 AAFE for the Area under the plasma concentration-time-profile curve (AUC) and maximal plasma concentration (C) predictions, respectively. Predicted C values were within ± 25% of observed values for 67% of all doses for the WT dogs. For the Mu dogs, the predicted AUC was within 50% for all doses. Our work provides the first example of a systematic approach for Collies and illustrates its use to describe the impact of a known genetic variation in the canine Mdr1 gene. Furthermore, we describe the general workflow for establishing, verifying, and applying an IVIVE-PBPK framework for predicting in vivo drug behavior within a specific canine breed.
Model-informed drug development (MIDD) methods play critical role to ensure development of efficacious, and safe individualized therapies. The application of artificial intelligence/machine learning (AI/ML) within the fi...Model-informed drug development (MIDD) methods play critical role to ensure development of efficacious, and safe individualized therapies. The application of artificial intelligence/machine learning (AI/ML) within the field of drug development has exponentially expanded. Integrating AI/ML into traditional pharmacometrics approaches or using AI/ML as a stand-alone tool has the potential to optimize dosing strategies, inform clinical trial designs, and enhance robustness of quantitative assessments of drug efficacy and safety. This review systematically evaluates the impact of AI-based model-informed drug development (MIDD) methods compared to traditional approaches by blending regulatory perspectives. We conducted a systematic search on PubMed using five Medical Subject Headings (MeSH) terms and included 67 relevant studies in the analysis. The results indicate that AI models have the potential of improving MIDD approaches through different stages of drug development to inform decision-making in clinical trials. However, limitations such as the lack of standardized evaluation metrics and standardized regulatory guidelines on the use of AI-based MIDD methods were noted. Overall, this review highlights the potential applications of AI in drug development and provides a foundation for future research to optimize and integrate AI-based approaches in this field.
Doxil®, a PEGylated liposomal doxorubicin (DOX) hydrochloride suspension, was the first liposome drug product approved by the U.S. Food and Drug Administration (FDA). Although off-patent, limited generic products have be...Doxil®, a PEGylated liposomal doxorubicin (DOX) hydrochloride suspension, was the first liposome drug product approved by the U.S. Food and Drug Administration (FDA). Although off-patent, limited generic products have been approved due to challenges in achieving bioequivalence compounded by manufacturing complexity. Regulatory agencies require generic drug products to be bioequivalent to the reference listed drug. In this regard, we developed various analytical methods to analyze Doxil® and its generic drug products for multiple attributes, including liposome size distribution, zeta potential, DOX content, lipid content, purity, non-encapsulated doxorubicin, morphology, nanostructure similarity, and quantified in vitro release. Batch-to-batch variation exists across attributes for different formulations. Minor differences in particle size and zeta potential were observed. Cryo-TEM imaging reveals the distinct coffee bean shape and morphology of the Doxil® liposome. SAXS similarity analysis shows a distinct difference in nanostructure for Dr. Reddy's formulation compared to the innovator formulation. Still, it is revealed to be a consequence of liposome uniformity and homogeneity as depicted in cryo-TEM images. Several methods developed in this work can be complementary to provide a more thorough physicochemical analysis for generic evaluation. Size, morphology, and nanostructure evaluated by DLS, cryo-TEM, and SAXS should be readily employed to assess physicochemical similarity. Content, impurity identification, and amount of free non-encapsulated DOX are used to evaluate formulation sameness. The methods developed in this work provide a physicochemical framework for analytical comparison of complex liposomal generics and may support subsequent development efforts to improve generic drug availability for patient populations in need.
Real-world data (RWD) and real-world evidence (RWE) are valuable resources for drug development strategies. Historically, it has been used for safety evaluation during post-marketing surveillance. RWD and RWE have been u...Real-world data (RWD) and real-world evidence (RWE) are valuable resources for drug development strategies. Historically, it has been used for safety evaluation during post-marketing surveillance. RWD and RWE have been utilized in the regulatory decision-making process for drug effectiveness, especially for rare diseases and cancers, where conducting randomized controlled trials is challenging. The Food and Drug Administration (FDA) is actively working on providing trustworthy information derived from RWD and RWE to supplement the data from clinical trials. This review discusses the potential use of RWE to make regulatory decisions on drug effectiveness for certain therapeutic areas as well as the challenges in drawing inferences on drug effectiveness from RWE. A review of FDA-approved new drug applications and biologics license applications suggests that several methodological considerations should be deliberated when designing a study using RWE to demonstrate product effectiveness. The acceptance of RWE, while promising, is dependent on the relevance and reliability of the data. The insight and engagement of all stakeholders contribute to the successful use of RWE for clinical evaluations.
Nitrosamines drug substance related impurities (NDSRI) are organic impurities, highly potent mutagenic substances that are classified as human carcinogens. Regulatory guidance's in this area provide procedures for contro...Nitrosamines drug substance related impurities (NDSRI) are organic impurities, highly potent mutagenic substances that are classified as human carcinogens. Regulatory guidance's in this area provide procedures for control and risk mitigation of NDSRI's in drug products. While efforts are made to control the NDSRI's, cases where NDSRI's are observed post- pivotal bioequivalence studies may require additional bioequivalence studies, between pre- and post- change formulations. The recent USFDA (United State Food and Drug Administration) guidance on "Control of Nitrosamine Impurities in Human Drugs" provides alternative BE methodologies for biopharmaceutics classification system (BCS) I, II and III drugs based on in vitro testing. For BCS IV molecule containing immediate release (IR) formulations, physiologically based pharmacokinetic (PBPK) modeling approach is recommended. In this context, this present article discusses use of PBPK modeling approaches as alternative BE methodologies for BCS IV molecule containing IR products. We have summarized use of in-house in silico tool for early risk prediction of NDSRI's solely based on molecule structure. This tool was used to predict the carcinogenic potential of 37 BCS IV molecules and further clinical exposure risk assessment was conducted on identified 5 high risk category molecules namely edoxaban, selumetinib, bosutinib, furosemide, hydrochlorothiazide where transporters are involved in absorption. The PBPK models were used to evaluate the impact of altered permeability and transporter kinetics on exposures, that may happen in presence of antioxidants. Further, the impact of permeability within ± 10-20% was evaluated on clinical exposures to determine permeability safe space (region within which bioequivalence is guaranteed as compared to the target formulation).
The selective delivery of small molecule compounds such as Gemcitabine to tumor cells is a promising methodology for enhancing therapeutic efficacy and attenuating the side effects of anticancer drugs. Aptamers are usefu...The selective delivery of small molecule compounds such as Gemcitabine to tumor cells is a promising methodology for enhancing therapeutic efficacy and attenuating the side effects of anticancer drugs. Aptamers are useful as target-directed ligands for tumor-selective drug delivery due to their ability to bind specific proteins. However, the drug must be released from the aptamer after the conjugate is taken up by the cell to exert its pharmacological effect. In this study, we designed and synthesized a conjugate in which a linker cleaved by glutathione, which is highly expressed in tumor cells, was inserted between the aptamer (AS1411) and Gemcitabine. Almost all Gemcitabine was released from the conjugate after 30 min in the presence of 6 mM glutathione. AS1411 is known to bind to nucleolin, which is highly expressed on tumor cells. The cytotoxicity of the AS1411 and Gemcitabine conjugate with a disulfide bond on A549 cells was higher than that of the conjugate without a disulfide bond. Furthermore, the cytotoxicity of the disulfide-linked conjugate of AS1411 and Gemcitabine was higher in A549 cells than in MCF10A cells, which were used as the model of normal cells. These results indicate that disulfide conjugation enhanced the tumor cell-selective cytotoxicity of Gemcitabine with AS1411.
Quality by Design (QbD) is a transformative and systematic approach to developing top-tier pharmaceutical products, ushering in a departure from traditional trial-and-error methods toward a more science-based, risk-orien...Quality by Design (QbD) is a transformative and systematic approach to developing top-tier pharmaceutical products, ushering in a departure from traditional trial-and-error methods toward a more science-based, risk-oriented, and holistic strategy. Central to QbD implementation is the meticulous development of formulations and manufacturing processes, consistently fulfilling predefined quality objectives. The core objective of QbD remains unwavering - to guarantee the steadfast alignment of the final pharmaceutical product with predetermined quality attributes, thereby mitigating batch-to-batch variations and potential recalls. This article succinctly explores the multifaceted application of QbD methodology within the pharmaceutical industry. Emphasizing its pivotal role in research and development, manufacturing, quality control, and quality assurance, the discussion navigates through the strategic deployment of QbD elements and tools. Amidst the evident advantages of QbD, challenges persist in its widespread adoption within the pharmaceutical sector and regulatory frameworks. This article sheds light on the regulatory landscape that currently governs the implementation of QbD in these crucial stages of pharmaceutical processes. For that reason, this review article aims to provide researchers, scientists, and industry professionals with a thorough introduction to QbD so they may adopt this methodical approach to developing and producing high-quality pharmaceutical products, always in compliance with the underlying regulations.
Accurate dose preparation and administration are critical components in clinical studies to ensure patient safety and drug efficacy. Closed System Transfer Devices (CSTD) are widely used for the preparation and administr...Accurate dose preparation and administration are critical components in clinical studies to ensure patient safety and drug efficacy. Closed System Transfer Devices (CSTD) are widely used for the preparation and administration of hazardous drugs. Recent literature has raised concerns about potential under- or over-dosing when doses are prepared using CSTD due to their high hold-up volume. To ensure dose accuracy, it is essential to follow proper preparation methods when using CSTD. Seven commonly used CSTD were evaluated for product transfer accuracy from vials to IV bags using four methods: no bag spike flushing, flushing bag spikes with the original syringes, flushing bag spikes with new syringes, and circle priming. The data confirmed that under- and over-dosing occurred frequently at transfer volume ≤ 2 mL when not flushing bag spikes or flushing bag spikes with the original syringes as suggested by some manufacturers' instructions. The study also demonstrated that flushing bag spikes with the new syringes or using circle priming is effective in enabling accurate volume transfer from vials to IV bags at transfer volumes ≤ 2 mL. Both methods are feasible for implementation in pharmacies or hospitals to ensure dose preparation accuracy using CSTD. It is recommended that clinical sites flush bag spikes with the new syringes or use circle priming method for low volume transfer to ensure dose preparation accuracy. CSTD manufacturers should consider including these methods in their Instructions for Use (IFU).
Administration of human therapeutic proteins such as monoclonal antibodies (mAb) to animals during preclinical drug development often leads to the development of anti-drug antibodies (ADA). ADA may reduce the systemic ex...Administration of human therapeutic proteins such as monoclonal antibodies (mAb) to animals during preclinical drug development often leads to the development of anti-drug antibodies (ADA). ADA may reduce the systemic exposure of the mAb by enhancing its immune-complex mediated clearance. Thus, ADA may hinder the preclinical pharmacokinetic and toxicology assessments of mAbs. To mitigate this effect, we explored the ability of short-term administration of immunosuppressants to induce prolonged immune tolerance towards a human mAb, erenumab, in rats. In two studies, we investigated dosing regimens using the immunosuppressants methotrexate and tacrolimus/sirolimus combination, and compared them to non-immunosuppressed control groups. Each study comprised three phases: induction (weeks 1-4), washout (weeks 5-8), and rechallenge (weeks 9-12). Animals received mAb during the induction and rechallenge phase, while immunosuppression was limited to the induction and washout phase. Blood samples were collected at predefined time-points for erenumab and ADA quantification. The tacrolimus/sirolimus regimen, but not the tested methotrexate regimens, completely prevented ADA formation in all treated animals relative to the control groups. The tacrolimus/sirolimus treated animals not only remained ADA-negative with initial immunosuppression during the induction phase but remained ADA-negative even after erenumab rechallenge suggesting the induction of immune-tolerance beyond the immunosuppressive treatment period. Correspondingly, erenumab systemic exposures were maintained throughout the study period in all animals in the tacrolimus/sirolimus group and were similar to the erenumab exposures in ADA-negative animals of the control group. In contrast, ADA-positive animals in the control group exhibited a 60-80% reduction in erenumab exposures.
The pharmacokinetics of antibodies with varied binding kinetics were simulated to assess the role of affinity and binding microconstants (kon, koff) on tumor exposure and intra-tumoral distribution. Anti-HER2 constructs...The pharmacokinetics of antibodies with varied binding kinetics were simulated to assess the role of affinity and binding microconstants (kon, koff) on tumor exposure and intra-tumoral distribution. Anti-HER2 constructs (trastuzumab, pertuzumab, VK3VH6, and conjugates with DM1 and gelonin) were produced, purified, and tested for binding and cytotoxicity in vitro, and for intra-tumoral distribution and anti-tumor efficacy in mice. Simulations demonstrated that homogeneity in intra-tumoral distribution increases with increases in koff and with decreases in kon. Interestingly, simulations also predicted that homogeneity in tumor distribution may be improved by decreasing kon and koff in parallel (without changing affinity). Relative to trastuzumab, pertuzumab exhibits similar affinity but a ~ fivefold smaller kon and koff, while VK3VH6 exhibits a similar koff but a ~ 30-fold lower kon and affinity. Conjugate concentrations associated with 50% inhibition of cell proliferation (IC50s) were found to vary with affinity, where IC50 values were similar for pertuzumab and trastuzumab, and higher for VK3VH6. Consistent with model simulations, VK3VH6 and pertuzumab demonstrated more homogeneous tumor distribution than trastuzumab. Although treatment differences were not statistically significant, pertuzumab and VK3VH6 conjugates showed trends for increased survival time relative to mice treated with trastuzumab conjugates. Our simulation and experimental results demonstrate complex relationships between antibody-antigen binding kinetics, intratumoral distribution, and efficacy. The rate constant of association, kon, is an underappreciated determinant of intra-tumoral distribution; among high-affinity antibodies, those with lower values of kon may be expected to exhibit improved intra-tumoral distribution and, potentially, efficacy.
Extensive analytical and functional characterization of a biotherapeutic product is a regulatory requirement, more so for biosimilar products where approval is contingent on the manufacturer's ability to demonstrate comp...Extensive analytical and functional characterization of a biotherapeutic product is a regulatory requirement, more so for biosimilar products where approval is contingent on the manufacturer's ability to demonstrate comparability of their product to the corresponding reference product. Typical biotherapeutic formulations contain multiple excipients that are meant to stabilize the product and these can impact certain analytical and functional techniques that are typically used in the above-mentioned characterization and comparability exercises. In this study, we elucidate this interference using Trastuzumab (Tmab) reference product, Herclon, and its commercially available biosimilars, Herzuma and Vivitra, as an example. Excipients were removed one at a time from the drug product and impact of this removal on a spectrum of analytical and functional tools was examined. Removal of certain excipients (Trehalose, L-histidine HCl and Polysorbate 20) was found to impact the results of charge variant analysis (cation exchange HPLC), secondary structure analysis (FTIR and far-UV CD spectroscopy), and tertiary structure analysis (near-UV CD and intrinsic FLR spectroscopy) For charge variants, differences up to 3.62% in basic species were observed, while FTIR spectra in the amide I region were significantly impacted. The intrinsic fluorescence spectra displayed major wavelength maxima shifts of up to 6 nm. In view of these results, it is recommended that biosimilar manufacturers consider the impact of differences in formulation in the samples that are being compared as well as the impact of excipient removal, if they are extracting the therapeutic moiety from the drug product for comparability analysis (routinely done by biosimilar manufacturers).
The purpose of this study was to establish a clinically relevant specification for carbamazepine (CBZ) tablets, a classic narrow therapeutic index drug (NTID), within the Chinese population. By integrating physiologicall...The purpose of this study was to establish a clinically relevant specification for carbamazepine (CBZ) tablets, a classic narrow therapeutic index drug (NTID), within the Chinese population. By integrating physiologically based pharmacokinetic (PBPK) modeling with in vitro dissolution profiles and corresponding pharmacokinetic (PK) data, we developed an in vitro-in vivo relationship (IVIVR) by selecting an appropriate dissolution model, and the IVIVR model was validated using in vitro and in vivo data from external sources to ensure the reliability of the method. Parameter sensitivity analysis was used to examine how the critical parameters influence the drug's absorption fraction (F). Additionally, the sensitivity of T, C, and AUC to physiological and formulation parameters was quantitatively evaluated. Based on the validated model, we also developed and validated a virtual bioequivalence (VBE) approach. Additionally, the safety space of the dissolution (Q ≥ 80% meanwhile 50% ≤ Q ≤ 85% and the assay (95 ~ 105%) for carbamazepine tablets (100 mg) were successfully explored depending on the VBE study. This study provides a valuable reference for establishing clinically relevant specifications for NTIDs through PBPK model-informed research.
Developing a mathematical model to predict the distribution and bioavailability of atropine in human eyes is an insight approach for clinical practice. This study aims to develop a human ocular physiologically based phar...Developing a mathematical model to predict the distribution and bioavailability of atropine in human eyes is an insight approach for clinical practice. This study aims to develop a human ocular physiologically based pharmacokinetic (PBPK) modeling for the ophthalmic drug atropine and explore possible mechanisms by which atropine reduces myopia progression in children. The Ocular Compartment Absorption and Transit (OCAT™) model was employed to describe the ocular distribution of atropine following administration at different dosages in both rabbits and humans. The PBPK model enables the extrapolation of pharmacokinetic characteristics among different species depending on theirphysiology and anatomy. The developed and validated OCAT-PBPK model demonstrated good agreement with observed data from rabbit ocular tissues and human aqueous humor. Fifty-eight percent of simulations fell within the standard deviation range of experimental data. The extrapolated human PBPK model for accurately predicted the ocular exposure and distribution following the administration of low-concentration atropine. This study confirms the performance of the ocular PBPK model in predicting ocular pharmacokinetic behavior among different species. Model's predictions indicate that atropine shows significant potential to penetrate the posterior eye segment, providing underlying insights into its mechanisms of action in the eye.
During drug development, the design, interpretation and risk assessment of drug-drug interaction (DDI) are generally performed with physiologically-based pharmacokinetic (PBPK) modelling. Critical parameters are the hepa...During drug development, the design, interpretation and risk assessment of drug-drug interaction (DDI) are generally performed with physiologically-based pharmacokinetic (PBPK) modelling. Critical parameters are the hepatic metabolic fraction (fm) and intestinal availability (F) which are commonly informed by clinical data. In this study, two methods for the simultaneous estimation of these parameters are proposed which utilize the distinctive changes in substrate's plasma concentration profiles in response to inhibition of intestinal and hepatic enzymes. The two-dimensional DDI (2D-DDI) method estimates the fm and F values directly from the ratios of area-under-curve (AUCR) and maximum concentration (CR), while the population PBPK method utilizes the full concentration-time data of a substrate without or with an inhibitor. The utility of both methods was demonstrated for a broad range of > 50,000 virtual and six actual CYP3A substrates. The 2D-DDI method is fast, reliable, and does not require a priori PBPK model development. The population PBPK method can estimate the population parameters and inter-individual variabilities of fm and F and is applicable to more complex DDIs (e.g., multiple pathways/dynamic inhibitor concentration-time profiles) without the need for IV data. Like other approaches, both methods show an increasing uncertainty for substrates with high hepatic extraction and sensitivity to the assumed degree of enzyme inhibition. While both methods were evaluated for CYP3A substrates, the methodology equally applies to other enzymes. Additionally, this study provides guidance for clinical DDI study design to facilitate robust DDI extrapolation necessary to inform drug labels on concomitant medications in lieu of clinical trials.