Tipifarnib physiologically-based pharmacokinetic modeling to assess drug-drug interaction, organ impairment, and biopharmaceutics in healthy subjects and cancer patients.

A physiologically-based pharmacokinetic (PBPK) model for tipifarnib, which included mechanistic absorption, was built and verified by integrating in vitro data and several clinical data in healthy subjects and cancer patients. The final PBPK model was able to recover the clinically observed single and multiple-dose plasma concentrations of tipifarnib in healthy subjects and cancer patients under several dosing conditions, such as co-administration with a strong CYP3A4 inhibitor and inducer, an acid-reducing agent (proton pump inhibitor and H2 receptor antagonist), and with a high-fat meal. In addition, the model was able to accurately predict the effect of mild or moderate hepatic impairment on tipifarnib exposure. The appropriately verified model was applied to prospectively simulate the liability of tipifarnib as a victim of CYP3A4 enzyme-based drug-drug interactions (DDIs) with a moderate inhibitor and inducer as well as tipifarnib as a perpetrator of DDIs with sensitive substrates of CYP3A4, CYP2B6, CYP2D6, CYP2C9, and CYP2C19 in healthy subjects and cancer patients. The effect of a high-fat meal, acid-reducing agent, and formulation change at the therapeutic dose was simulated. Finally, the model was used to predict the effect of mild, moderate, or severe hepatic, and renal impairment on tipifarnib PK. This multipronged approach of combining the available clinical data with PBPK modeling-guided dosing recommendations for tipifarnib under several conditions. This example showcases the totality of the data approach to gain a more thorough understanding of clinical pharmacology and biopharmaceutic properties of oncology drugs in development.

Rare oncology therapeutics: review of clinical pharmacology package of drug approvals (2019-2023) by US FDA, best practices and recommendations.

There are many challenges with rare diseases drug development and rare oncology indications are not different. To understand the regulatory landscape as it relates to application of clinical pharmacology principles in rare oncology product development, we reviewed publicly available information of 39 approvals by US FDA between January 2019 and March 2023. The objective was to understand the expected clinical pharmacology studies and knowledge base in such approvals. Model informed drug development (MIDD) applications were also reviewed, as such approaches are expected to play a critical role in filling clinical pharmacology gaps in rare oncology, where number of clinical trials and size of these trials will perhaps continue to be small. The findings highlighted how clinical pharmacology contributed to the evidence of effectiveness, dose optimization and elucidation of intrinsic and extrinsic factors affecting drug's behavior. Clinical pharmacology studies were often integrated with modeling in many of the NDAs/BLAs. Of the post marketing requirements (PMR) received, 18% were for dose optimization, 49% for DDI, 8% for QTc, 49% for specific population, and 5% for food effect. Two post marketing commitments (PMC) were issued for immunogenicity of the 11 biologics submissions. 15% (6 of 39) of the submissions used maximum tolerated dose (MTD) to advance their molecule into Phase 2 studies. Of them 3 approvals received PMR for dose optimization. 3 + 3 was the most prevalent Phase 1 design with use in 74% of the New Drug Applications (NDA)/Biologic License Applications (BLA) reviewed. Rest used innovative approaches such as BLRM, BOIN or mTPi, with BLRM being the most common. Seamless clinical pharmacology and MIDD approaches are paramount for rare oncology drug development.

Model-Informed Approaches and Innovative Clinical Trial Design for Adeno-Associated Viral Vector-Based Gene Therapy Product Development: A White Paper.

The promise of viral vector-based gene therapy (GT) as a transformative paradigm for treating severely debilitating and life-threatening diseases is slowly coming to fruition with the recent approval of several drug products. However, they have a unique mechanism of action often necessitating a tortuous clinical development plan. Expertise in such complex therapeutic modality is still fairly limited in this emerging class of adeno-associated virus (AAV) vector-based gene therapies. Because of the irreversible mode of action and incomplete understanding of genotype-phenotype relationship and disease progression in rare diseases careful considerations should be given to GT product's benefit-risk profile. In particular, special attention needs to be paid to safe dose selection, reliable dose exposure response (including clinically relevant endpoints), or creative approaches in study design targeting small patient populations during clinical development. We believe that quantitative tools encompassed within model-informed drug development (MIDD) framework fits quite well in the development of such novel therapies, as they enable us to benefit from the totality of data approach in order to support dose selection as well as optimize clinical trial designs, end point selection, and patient enrichment. In this thought leadership paper, we provide our collective experiences, identify challenges, and suggest areas of improvement in applications of modeling and innovative trial design in development of AAV-based GT products and reflect on the challenges and opportunities for incorporating MIDD tools and more in rational development of these products.

Impact of gastrointestinal tract variability on oral drug absorption and pharmacokinetics: An UNGAP review.

The absorption of oral drugs is frequently plagued by significant variability with potentially serious therapeutic consequences. The source of variability can be traced back to interindividual variability in physiology, differences in special populations (age- and disease-dependent), drug and formulation properties, or food-drug interactions. Clinical evidence for the impact of some of these factors on drug pharmacokinetic variability is mounting: e.g. gastric pH and emptying time, small intestinal fluid properties, differences in pediatrics and the elderly, and surgical changes in gastrointestinal anatomy. However, the link of colonic factors variability (transit time, fluid composition, microbiome), sex differences (male vs. female) and gut-related diseases (chronic constipation, anorexia and cachexia) to drug absorption variability has not been firmly established yet. At the same time, a way to decrease oral drug pharmacokinetic variability is provided by the pharmaceutical industry: clinical evidence suggests that formulation approaches employed during drug development can decrease the variability in oral exposure. This review outlines the main drivers of oral drug exposure variability and potential approaches to overcome them, while highlighting existing knowledge gaps and guiding future studies in this area.

Design of Controlled Release PLGA Microspheres for Hydrophobic Fenretinide.

Fenretinide, a chemotherapeutic agent for cancer, is water-insoluble and has a very low oral bioavailability. Hence, the objective was to deliver it as an injectable depot and improve the drug solubility and release behavior from poly(lactide-co-glycolide) (PLGA) microspheres by incorporating nonionic surfactants with fenretinide. Enhancement of drug solubilization was observed with Brij 35 or 98, Tween 20, and Pluronic F127, but not Pluronic F68. Co-incorporation of Brij 98 with fenretinide significantly changed the microsphere morphology and improved the fenretinide release profile. The most optimal microsphere formulation, with 20% Brij 98 as excipient, showed an initial in vitro burst around 20% and a sustained release over 28 days in a solubilizing release medium at 37 °C. The effect of addition of MgCO3, drug loading, and polymer blending on the release of fenretinide from PLGA microspheres was also investigated and observed to enhance the drug release. Two sustained release formulations, one incorporating 20% Brij 98 and the other incorporating 3% MgCO3 in the oil phase, were selected for dosing in Sprague-Dawley rats and compared to a single injection of an equivalent dose of fenretinide drug suspension. These two formulations were chosen due to their high encapsulation efficiency, high cumulative release, and desirable in vitro release profile. The drug suspension resulted in a higher initial release in rats compared to the polymeric formulations, however, sustained release was also observed beyond 2 weeks, which may be attributed to the physiological disposition of the drug in vivo. The two PLGA based test formulations provided the desired low initial burst of fenretinide followed by 4 weeks of in vivo sustained release.

Use of minipig skin biopsy model as an innovative tool to design topical formulation to achieve desired pharmacokinetics in humans.

In vitro cadaver skin permeation studies are often conducted to characterize the permeation profile of compounds for dermal delivery. However, its utility could be limited in the case of topical products because of lack of reliable prediction of in vivo skin kinetics. In this paper, the use of in vivo skin biopsy data to guide topical formulation development is described. A formulation was developed by compounding MK-0873, a phosphodiesterase 4 (PDE4) inhibitor, into a commercially available cream base. The cream was characterized by skin pharmacokinetic studies in minipigs, which demonstrated that MK-0873 concentrations in the epidermis and dermis were substantially higher than the IC80 for human whole blood PDE4 inhibition of ∼200 nM, suggesting that cream should provide sufficient skin exposure to assess clinical efficacy. In toxicological studies, after 1 month repeat application in minipigs minor dermal irritation and minimal systemic exposure were observed. Based on these preclinical data, the cream formulation was chosen for single rising dose clinical studies, where plasma levels of MK-0873 were mostly below the LOQ, whereas skin biopsy concentrations ranged from 6.5 to 25.1 µM. These data suggested that minipig skin biopsy model can be a valuable tool to assess performance of topical formulations and guide formulation development.

Lactoferrin-derived resistance against plant pathogens in transgenic plants.

Lactoferrin (LF) is a ubiquitous cationic iron-binding milk glycoprotein that contributes to nutrition and exerts a broad-spectrum primary defense against bacteria, fungi, protozoa, and viruses in mammals. These qualities make lactoferrin protein and its antimicrobial motifs highly desirable candidates to be incorporated in plants to impart broad-based resistance against plant pathogens or to economically produce them in bulk quantities for pharmaceutical and nutritional purposes. This study introduced bovine LF (BLF) gene into tobacco ( Nicotiana tabacum var. Xanthi), Arabidopsis ( A. thaliana ) and wheat ( Triticum aestivum ) via Agrobacterium -mediated plant transformation. Transgenic plants or detached leaves exhibited high levels of resistance against the damping-off causing fungal pathogen Rhizoctonia solani and the head blight causing fungal pathogen Fusarium graminearum . LF also imparted resistance to tomato plants against a bacterial pathogen, Ralstonia solanacearum . Similarly, other researchers demonstrated expression of LF and LF-mediated high-quality resistance to several other aggressive fungal and bacterial plant pathogens in transgenic plants and against viral pathogens by foliar applications of LF or its derivatives. Taken together, these studies demonstrated the effectiveness of LF for improving crop quality and its biopharming potentials for pharmaceautical and nutritional applications.

The intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes functions as a bidirectional promoter.

Genetic engineering plays a unique role in fundamental plant biology studies and in improving crop traits. These efforts often necessitate introduction and expression of multiple genes using promoters from a very limited repertoire. Current common practice of expressing multiple genes is the repeated use of the same or similar promoters. This practice causes more frequent transgene silencing due to a high degree of sequence homology and a greater chance of rearrangement among repeatedly used promoter sequences. Therefore, availability and use of natural bidirectional promoters to minimize gene silencing and achieve desirable expression pattern of transgenes is a critical issue in the field of plant genetic engineering. Here we describe the use of a single natural bidirectional promoter to drive the expression of two reporter genes in onion epidermal cells and in transgenic tobacco plants. We show that (1) the promoter drives the simultaneous expression of GUS and GFP reporter genes after transient expression and stable transformation, (2) the transcription is equally strong in both directions, (3) immediate upstream regions in each direction control transcription independently from each other, and (4) the reporter genes are expressed in leaves and stems but not in roots, as expected from the fact that the endogenous promoter controls the expression of two photosynthetic genes in Arabidopsis. Hence, use of bidirectional promoters in heterologous background provides a means to express multiple genes in transgenic plants and aids genetic engineering-based crop improvement.

Nanocarriers for nuclear imaging and radiotherapy of cancer.

Several nanoscale carriers (nanoparticles, liposomes, water-soluble polymers, micelles and dendrimers) have been developed for targeted delivery of cancer diagnostic and therapeutic agents. These carriers can selectively target cancer sites and carry large payloads, thereby improving cancer detection and therapy effectiveness. Further, the combination of newer nuclear imaging techniques providing high sensitivity and spatial resolution such as dual modality imaging with positron emission tomography/computed tomography (PET/CT) and use of nanoscale devices to carry diagnostic and therapeutic radionuclides with high target specificity can enable more accurate detection, staging and therapy planning of cancer. The successful clinical applications of radiolabeled monoclonal antibodies for cancer detection and therapy bode well for the future of nanoscale carrier systems in clinical oncology. Several radiolabeled multifunctional nanocarriers have been effective in detecting and treating cancer in animal models. Nonetheless, further preclinical, clinical and long-term toxicity studies will be required to translate this technology to the care of patients with cancer. The objective of this review is to present a brief but comprehensive overview of the various nuclear imaging techniques and the use of nanocarriers to deliver radionuclides for the diagnosis and therapy of cancer.

Polymeric conjugates of mono- and bi-cyclic alphaVbeta3 binding peptides for tumor targeting.

The alphaVbeta3 integrin plays important roles in tumor-induced angiogenesis and tumor metastasis and hence, many small molecule alphaVbeta3 ligands have been developed for cancer diagnosis and therapy. Although these show good alphaVbeta3 targeting, most have suboptimal pharmacokinetics and show rapid tumor washout. We studied the biodistribution and tumor targeting properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based conjugates of mono-(RGDfK) and doubly cyclized (RGD4C) alphaVbeta3 binding peptides. Endothelial cell adhesion studies showed similar affinity of HPMA-RGD4C and HPMA-RGDfK conjugate for alphaVbeta3 integrins. Scintigraphic images of tumor bearing mice demonstrated that both conjugates showed tumor localization at 24 h post-injection and were retained at the tumor site until 192 h, whereas the efficient background clearance was observed over time. Necropsy organ counts showed that tumor accumulation of both HPMA-RGD4C and HPMA-RGDfK conjugates increased over time with peak accumulations at 4.9 +/- 0.9% and 5.0 +/- 1.2% ID/g, respectively. In contrast the background organ distribution rapidly cleared over time resulting in significant increases of tumor-to-background ratios. The radioactive dose as indicated by the area under curve (HPMA-RGD4C: 4825.3 microCi/g h and HPMA-RGDfK: 4424.9 microCi/g h) was highest for the tumor. The polymer conjugates of RGD4C or RGDfK provide a means to enhance tumor uptake, decrease background accumulation, and enable selective delivery of therapeutic or diagnostic agents to tumor sites.

Polymer-peptide conjugates for angiogenesis targeted tumor radiotherapy.

INTRODUCTION: New methods of delivering radiotherapy to sites of occult or disseminated cancer are needed to control the disease and address the failure of conventional therapy. Because tumor cells rely on angiogenesis for survival, we assessed the effectiveness of beta-emitter radiotherapy delivered by polymer-peptide conjugates that target tumor neovasculature. This molecularly targeted radiation is intended to damage both the endothelial bed and surrounding neoplastic cells. METHODS: N-(2-Hydroxypropyl) methacrylamide (HPMA), a biocompatible and water-soluble copolymer, was derivatized to incorporate side chains for (99m)Tc and (90)Y chelation and was further conjugated to a alpha(V)beta(3) integrin-targeting peptide (RGD4C). The HPMA copolymer-RGD4C conjugate was characterized by its side-chain contents, in vitro endothelial cell adhesion assay and its biodistribution and antitumor effectiveness in a SCID mouse xenograft model of human prostate carcinoma. RESULTS: The conjugate contained about 16 RGD4C moieties per polymer backbone. Tumor accumulation significantly increased (P < .01) over time from 1.05 +/- 0.03 % injected dose (%ID)/g tissue at 1 h to 4.32 +/-0.32% at 72 h. The activity in major normal tissues significantly decreased (P < .05) during that period. At 21 days, the control tumors increased 442% in volume from baseline. In contrast, a 7% and a 63% decrease of tumor volume were observed for the 100- and 250-microCi (90)Y treatment groups, respectively. Histopathological examination revealed increased apoptosis in the treated tumors with no acute signs of radiation-induced toxicity to other organs. CONCLUSION: This copolymer-peptide conjugate targets tumor angiogenic vessels and delivers sufficient radiotherapy to arrest tumor growth.

Targeting tumor angiogenesis: comparison of peptide and polymer-peptide conjugates.

UNLABELLED: Endothelial cells in tumor angiogenesis are highly accessible, genetically stable and present unique molecular markers for targeted therapy. Neoplasia is also characterized by enhanced vascular permeability and disordered lymphatics so that both active and passive targeting strategies may play a role in localizing angiogenesis-targeted agents. To investigate the relative importance of these targeting strategies, the tissue biodistribution of both endothelial-specific and nonspecific peptides and their macromolecular peptide-copolymer conjugates were studied in 2 xenograft models of prostate cancer. Tumor-to-normal tissue background ratios (T/B) of these constructs were compared to evaluate the effect of molecular size on blood clearance and nonspecific vascular permeability. METHODS: Water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized with side chains terminated in a doubly cyclized Arg-Gly-Asp motif KACDCRGDCFCG (RGD4C: active peptide targeting the alpha(V)beta(3) integrin) and KACDCRGECFCG (RGE4C: nonactive peptide). The bioactivity of the polymer conjugates and free peptides was characterized in vitro by endothelial cell adhesion assay. The (99m)Tc(CO)(3)-labeled compounds were injected into SCID mice bearing DU145 or PC-3 prostate tumor xenografts for scintigraphic imaging and necropsy organ counting. RESULTS: HPMA copolymer-RGD4C conjugates showed similar inhibition of cell adhesion as free RGD4C attached to (99m)Tc(CO)(3) chelator N-omega-bis(2-pyridylmethyl)-L-lysine (RGD4C-DPK) and were significantly higher (P < 0.05) than RGE4C, HPMA copolymer-RGE4C, and a hydrolyzed HPMA copolymer precursor. Scintigraphic images obtained at 24 h showed specific tumor localization of HPMA copolymer-RGD4C and RGD4C compared with RGE4C conjugates in both prostate tumor models. Twenty-four-hour necropsy data in the DU145 model showed significantly higher (P < 0.001) tumor localization for HPMA copolymer-RGD4C (4.60 +/- 1.80%ID/g [percentage injected dose per gram tissue]) and RGD4C-DPK (3.37 +/- 0.32%ID/g) compared with HPMA copolymer-RGE4C (1.24 +/- 0.15%ID/g) and RGE4C-DPK (0.32 +/- 0.04%ID/g). Similar results were observed in the PC-3 model. Moreover, higher T/B for the polymer conjugates indicated reduced extravasation of the targeted polymeric conjugates in normal tissues. CONCLUSION: Specific molecular targeting of the alpha(v)beta(3) integrin and nonspecific vascular permeability are both significant in the relative tumor localization of polymeric conjugates of RGD4C. Extravascular leak in nonspecific organs appears to be a major factor in reducing the T/B for the peptide molecules.

Targeting tumor angiogenic vasculature using polymer-RGD conjugates.

Sites of neovascular angiogenesis are important chemotherapy targets. In this study, the synthesis, characterization, in-vivo imaging and biodistribution of a technetium-99m labeled, water-soluble, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer carrying doubly cyclized Arg-Gly-Asp motifs (HPMA copolymer-RGD4C conjugate) are reported. In vitro endothelial cell adhesion assays indicated that HPMA copolymer-RGD4C conjugates inhibited alphaVbeta3-mediated endothelial cell adhesion while HPMA copolymer Arg-Gly-Glu control conjugates (HPMA copolymer-RGE4C conjugate) and hydrolyzed HPMA copolymer precursor (HPMA copolymer) showed no activity. The scintigraphic images of prostate tumor bearing SCID mice obtained 24 h post-i.v. injection indicated greater tumor localization of HPMA copolymer-RGD4C conjugate than the control, HPMA copolymer-RGE4C conjugate. The 24-h necropsy radioactivity data showed that HPMA copolymer-RGD4C conjugate had significantly higher (p<0.001) tumor localization compared to HPMA copolymer-RGE4C conjugate and HPMA copolymer. Also, HPMA copolymer-RGD4C conjugates had sustained tumor retention over 72 h and reasonably efficient clearance from the background organs. These results suggest that specific tumor angiogenesis targeting is possible with HPMA copolymer-RGD4C conjugates. This construct provides a foundation that should support targeted delivery of radionuclides and drugs to solid tumors for diagnostic and therapeutic applications.

Technetium-99m-Labeled N-(2-hydroxypropyl) methacrylamide copolymers: synthesis, characterization, and in vivo biodistribution.

PURPOSE: To synthesize novel technetium-99m (99mTc)-labeled N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers and characterize the effect of charge and molecular weight on their biodistribution in SCID mice. METHODS: Electronegative and neutral 7-kDa, 21-kDa, and 70-kDa HPMA copolymers containing a 99mTc chelating comonomer, bearing N-omega-bis(2-pyridylmethyl)-L-lysine (DPK), were synthesized by free-radical precipitation copolymerization. The copolymers were labeled via 99mTc tricarbonyl chelation to DPK-bearing comonomer. They were characterized by side-chain content, molecular weight, molecular weight distribution, radiochemical purity, and labeling stability. Scintigraphic images were obtained during the first 90 min and at 24 h postintravenous injection in SCID mice. At 24 h, organ radioactivity was determined from necropsy tissue counting. RESULTS: 99mTc-labeled HPMA copolymers showed greater than 90% stability over a 24-h challenge with cysteine and histidine. Scintigraphic images and the necropsy data showed that the negatively charged copolymers were eliminated from the body significantly faster than the neutral copolymers in a size-dependent manner. CONCLUSIONS: To facilitate clinical scintigraphic imaging, stable chelation of 99mTc may be achieved by incorporation of a DPK-bearing comonomer into the HPMA backbone. Electronegative and neutral 99mTc-labeled HPMA copolymers of 7, 21, and 70 kDa show significant variation in organ biodistribution in SCID mice. 99mTc-labeled HPMA copolymers could be used as diagnostic agents and to study pharmacokinetics of delivery systems based on these copolymers.

Effect of surfactant on fabrication and characterization of paclitaxel-loaded polybutylcyanoacrylate nanoparticulate delivery systems.

The feasibility of applying biodegradable polybutylcyanoacrylate (PBCA) nanoparticulate delivery systems (NDSs) for the controlled release of paclitaxel was investigated. Paclitaxel-loaded and unloaded PBCA-NDSs containing various surfactants (dextran 70, cholesterol, polyvinyl alcohol and lecithin) were prepared by anionic polymerization. The effects of surfactant (1% w/v), surfactant combination (1% w/v each), and surfactant concentration (0.05, 1.0 and 2.5% w/v) on PBCA-NDSs were evaluated and characterized by particle size, zeta potential, entrapment efficiency, and in-vitro paclitaxel release kinetics. The physicochemical characteristics of PBCA-NDSs incorporated with various surfactants were significantly improved compared with PBCA-NDS without any surfactant, by decreasing particle size at least 3-fold as well as by increasing the zeta potential up to 18-fold to minimize the agglomeration of nanoparticles. Moreover, PBCA-NDSs incorporated with various surfactants demonstrated higher entrapment efficiency of paclitaxel. Results from the in-vitro release kinetic studies indicated that a more controlled biphasic zero-order release pattern of paclitaxel was observed for PBCA-NDSs incorporated with various surfactants. Compared with dextran 70 and polyvinyl alcohol, the naturally occurring lipids, lecithin and cholesterol, indicated greater advantages in improving the physicochemical properties of PBCA-NDSs, in terms of smaller particle size, higher zeta potential and better drug entrapment efficiency, and better controlled release of paclitaxel, in terms of lower release rate and prolonged action from PBCA-NDSs.

Intrinsic direct repeats generate consistent post-transcriptional gene silencing in tobacco.

It is well documented that transgenes with inverted repeats can efficiently trigger post-transcriptional gene silencing (PTGS), presumably via a double stranded RNA induced by complementary sequences in their transcripts. We show here that transgenes with intrinsic direct repeats can also induce PTGS at a very high frequency (80-100%). A transgene with three or four repeats induced PTGS in almost 100% of the primary transformants, regardless of whether a strong (enhanced 35S promoter) or a relatively weak (chlorophyll a/b binding protein promoter) promoter was used. The PTGS induced by three or four repeats is consistently inherited in subsequent generations, and can inactivate homologous genes in trans. Based on the high frequency and consistent heritability, we propose that the intrinsic direct repeat within a transgene may act as a primary determinant of PTGS referred to as direct repeat-induced PTGS (driPTGS). Silencing occurred in all five genes, in this and two previous reports, suggesting that driPTGS might be a universal gene silencing mechanism both in dicotyledonous tobacco plants and monocotyledonous rice cells. In addition, driPTGS may help dissect the gene silencing mechanism and generate silenced phenotypes useful for research and plant biotechnology products.