Molecular Pharmaceutics最新文献

Folate receptor alpha(FRα) is frequently overexpressedinovarian cancer while showing limited expression in healthy tissues.Targeting of FRα has been extensively investigated using antibody-basedtherapies but less so with small scaffold proteins. Small scaffoldproteins represent an alternative approach with potential advantagessuch as site-specific conjugation and deep tumor penetration but arelimited by a short half-life. Here, we describe the development ofa dual-surfaced ADAPT scaffold protein (6 kDa) with simultaneous bispecificityfor FRα and albumin. Using combinatorial libraries and phagedisplay, we identified a candidate, termed ADAPT22, with high isoformselectivity and subnanomolar affinity for FRα (K_D = 0.8 nM), paired with a low nanomolar affinity foralbumin (K_D = 2.4 nM). Importantly, ADAPT22is able to interact with both targets simultaneously, truly combiningextended retention in blood via albumin association and tumor targetingvia FRα within a single-domain protein. The simultaneous naturewas verified in vitro to soluble proteins and toFRα-positive cells and in vivo using a mousexenograft model. In mice, ADAPT22 demonstrated markedly extended bloodretention, reduced renal uptake, and specific accumulation in FRα-positivetumors, confirming simultaneous dual-surface binding. To the bestof our knowledge, this is the smallest half-life extended affinityprotein designed for therapeutic targeting of FRα.

Itraconazole (ITR)and posaconazole (POS) exhibit closely relatedmolecular structures but differ significantly in their ability toform liquid crystalline (LC) phases. This makes them an attractivemodel system to investigate how subtle structural differences influencemolecular organization in the amorphous state. In this work, we investigatecoamorphous systems composed of ITR and POS prepared via melt-quenching,focusing on the interplay between molecular dynamics, physical stability,and LC ordering. Differential scanning calorimetry (DSC) and broadbanddielectric spectroscopy (BDS) reveal that the glass transition temperatureremains nearly unchanged over a wide compositional range, indicatingcomparable molecular mobility of all investigated systems. Despitethis similarity, a pronounced, nonmonotonic dependence of crystallizationkinetics on composition is observed, with a clear stability maximumat the eutectic ratio. Strikingly, this specific composition is alsoassociated with the complete disappearance of the LC ordering characteristicof neat ITR. While ITR-rich mixtures close to the eutectic point stillexhibit residual LC transitions, the eutectic composition yields afully disordered amorphous phase. These results demonstrate, for thefirst time, that coamorphization at the eutectic composition mightsuppress LC ordering. The findings highlight the critical role ofcomposition in governing both molecular organization and stabilityin amorphous pharmaceuticals.

Nectin-4–targetedimaging has emerged as a promising strategyfor patient stratification in urothelial carcinoma, yet currentlyavailable radiotracers remain limited by rapid renal clearance, urinarybackground activity, or potential pharmacologic confounding associatedwith antibody-drug conjugate (ADC)-based constructs. To overcome someof these limitations, we developed a payload-free immunoPET radiotracerbased on the native enfortumab antibody conjugated with the bifunctionalchelator DFO*-sq and radiolabeled with zirconium-89 (⁸⁹Zr). The resulting [⁸⁹Zr]Zr-DFO*-sq-Enfortumab preservedNectin-4–specific targeting while demonstrating high radiochemicalstability in human serum (>99% through Day 12). In vitro studies showed efficient antigen-specific cellular internalization(80–85%), prolonged intracellular retention, and evidence consistentwith partial recycling of antibody-associated species. In vivo, [⁸⁹Zr]Zr-DFO*-sq-Enfortumab exhibited sustainedtumor uptake reaching 17.4 ± 2.8% ID/g in high Nectin-4–expressingxenografts and correlated with differential Nectin-4 expression acrossmultiple bladder cancer models. Importantly, the full-length antibodyplatform enabled high-contrast visualization of pulmonary metastaticlesions and orthotopic bladder tumors despite some urinary backgroundactivity. Together, these findings demonstrate that payload-free [⁸⁹Zr]Zr-DFO*-sq-Enfortumab provides a biologically relevantimmunoPET platform for noninvasive assessment of variable Nectin-4expression and support its further evaluation as a companion imagingstrategy for Enfortumab Vedotin–based therapies.

Driven by the evolving demands ofmodern pharmaceutical industry,high-concentration monoclonal antibody formulations (HCMFs) have emergedas a central research hotspot in biopharmaceutical development, attributableto their merits such as low administration volume and reduced medicalexpenditure. For lyophilized HCMFs, reconstitution performance servesas a critical quality attribute of equivalent importance to long-termstorage stability. As components in direct contact with lyophilizeddrug products, primary packaging containers are indispensable criticalexcipients for pharmaceutical preparation. This work systematicallyinvestigated the effects of inner surface characteristics of glassvials on the long-term stability and reconstitution behaviors of lyophilizedHCMFs. The results demonstrated that the surface hydrophilicity/hydrophobicityof vial walls exerted no remarkable influence on the physicochemicalstability of mAb-X during prolonged storage. Nevertheless, in thereconstitution process, the intrinsically high viscosity of concentratedmAb solutions, combined with the ultralow surface free energy, intensehydrophobicity and weakened contact line pinning effect of such vials,triggered massive visible-bubbles to adhere beneath the liquid–solidinterface. These trapped bubbles failed to ascend and dissipate alongthe inner walls and bottom of vials. Collectively, this study verifiesthat hydrophobic primary packaging materials, when adopted as storagecontainers for lyophilized HCMFs, will induce severe reconstitutiondefects, which may further lead to inaccurate dosing and compromisedclinical operability.

The gastrin-releasing peptide receptor (GRPR) is an attractivetarget for breast cancer (BC) imaging, but the in vivo performance of GRPR-targeted radioligands, such as RM2 and NeoB,is limited by metabolic instability, which may compromise tumor retention.Here, we designed two dimeric RM2-based radioligands, hdPP-01 andhdPP-02, to investigate whether homodimerization could improve thepreclinical profile of GRPR-targeted radiotracers. hdPP-01 was synthesizedusing a 1,3,5-triazine core, whereas hdPP-02 was obtained via a 2-cyanobenzothiazole/1,2-aminothiolclick reaction. Both compounds were radiolabeled with indium-111 andevaluated in T47-D cells. The resulting radiotracers retained nanomolarGRPR-binding properties, showed higher GRPR-specific cellular uptake,and displayed improved stability in murine serum compared to [¹¹¹In]In-RM2. In vivo, [¹¹¹In]In-hdPP-01demonstrated higher tumor uptake and prolonged retention time relativeto [¹¹¹In]In-RM2. These findings indicate that homodimerizationcan modulate stability, cellular uptake and tumor accumulation, supportingfurther optimization of dimeric RM2-based radioligands for GRPR-targetedBC imaging and therapy.

Serodolin is a biased agonist of the 5-HT₇ receptorsthat has demonstrated promising antinociceptive properties in preclinicalmodels. To further support the development of this potential drug,we decided to use PET imaging to demonstrate in vivo the capacity of Serodolin to cross the blood–brain barrierand bind specifically in the brain. For that matter, Serodolin wasisotopically radiolabeled with carbon-11 (β⁺, t_1/2 = 20.4 min) using the Staudinger/aza-Wittigstrategy and an azide precursor synthesized in 3 steps with an 18%yield. After optimization, automated radiosynthesis afforded [¹¹C]Serodolin in 79 ± 4% decay-corrected radiochemicalyield (n = 6) within 30 min. Quality control performedby analytical HPLC showed that ready-to-inject [¹¹C]Serodolinwas obtained with over 95% radiochemical and chemical purities anda molar activity of 55 ± 3 GBq/μmol (n = 6). Blood kinetics of [¹¹C]Serodolin performed withand without coinjection of nonradioactive Serodolin (5 mg/kg) in healthyrats displayed a rapid distribution (1.25 min) and fast systemic clearancewith no significant difference between the tracer and pharmacologicaldoses. Brain PET imaging and kinetics in healthy rats showed a rapidaccumulation of [¹¹C]Serodolin (C_max = 1.70 ± 0.35 standard uptake value at 3 min postinjection)and a widespread distribution throughout the brain parenchyma, whereascoinjection with nonradioactive Serodolin (5 mg/kg) resulted in ageneralized and significant (p = 0.000012, two-wayANOVA) reduction of brain exposure (area under the curve (0–60)< 30%). These results demonstrate for the first time in vivo a reduced brain exposure under pharmacological coinjection,consistent with central target engagement of Serodolin, and encouragefurther development of this molecule as a potential antinociceptivedrug.

Polymeric excipients are generally used for physicalstabilizationof amorphous drugs. However, a more strategic design of amorphousformulations is needed to reduce the amount of excipients, for whichthe use of mesoporous materials can be a promising option. In thisstudy, mesoporous silica (MS) with various particle and pore sizeswere added to amorphous acetaminophen (AAP) at various mixing ratiosto systematically clarify their stabilization mechanisms. In particular,how the presence of MS affects the stability, at mixing ratios whereAAP is present in excess relative to the pore volume, was focused.Six types of MS, with pore and particle sizes ranging from 2.5 to21 nm and from 3 to 9 μm, respectively, were used in the study.Differential scanning calorimetry (DSC) analysis suggested the presenceof three molecular fractions of amorphous AAP with different molecularmobilities: free, intermediate, and rigid fractions. The free fractionbehaved similarly to pure AAP, whereas the intermediate fraction appearedto be relatively more stable. The rigid fraction had limited molecularmobility and was expected to strongly interact with the MS surface.AAP molecules mainly existed as the free fraction in the presenceof 10% MS, regardless of the MS type. The free fraction disappearedto allow domination by the intermediate fraction with 50% MS having17 or 21 nm pores, whereas a significant amount of the free fractionremained with MS having 2.5 nm pores. Broadband dielectric spectroscopy(BDS) analysis revealed that the addition of MS both increased anddecreased the molecular mobility of amorphous AAP. The presence ofall types of 10% MS slightly accelerated the γ-relaxation ofamorphous AAP without affecting the α-relaxation. The additionof 50% MS with 17 or 21 nm pores slowed down the a-relaxation time of amorphous AAP, whereas the effect was marginalfor MS with 2.5 nm pores. Isothermal crystallization of amorphousAAP at 60 °C was accelerated in the presence of MS with 2.5 nmpores at mixing ratios of both 10 and 50%. In contrast, 50% MS with17 or 21 nm pores exhibited a strong stabilization effect. The acceleratedcrystallization in the presence of MS with 2.5 nm pores was explainedby an increase in local molecular mobility. Mixtures with MS with17 or 21 nm pores exhibited higher stability, which was explainedby the large portion of the intermediate fraction with slower motionrelative to that of pure AAP. The intermediate fraction was assumedto move inside and outside the pores, and the ease of molecular exchangewas explained by the size of the pores and characteristic length ofthe cooperatively rearranging region of amorphous AAP. These observationsprovide guidance for the selection of MS materials for the stabilizationof large amounts of pharmaceutical glasses and the scientific significanceof the molecular dynamics of glasses in the presence of porous materials.

After intravenous administration, some heptamethine cyaninedyesbind to serum albumin and accumulate in tumors via the enhanced permeabilityand retention effect. Additionally, asymmetric cyanine dyes have twoindole moieties which allow conjugation of a low-molecular-weightanticancer agent to one of the N-substituent sidechains. Therefore, asymmetric heptamethine cyanine dyes have beenstudied as possible platforms for tumor-targeted drug delivery. However,the effects of functional groups on the opposite side chains on theirin vivo behavior have not been explored in detail. Therefore, in thisstudy, we aimed to clarify these effects by designing three modelasymmetric cyanine dyes. For each dye, the side chain opposite thedrug-conjugation site was modified with either an alkyl chain, a carboxylgroup, or a sulfonyl group. A ¹²⁵I atom was introducedon the other side of the molecule to enable quantitative biodistributionanalysis. The resulting compounds are referred to as [¹²⁵I]Bu, [¹²⁵I]COOH, and [¹²⁵I]SO₃H.The albumin binding affinities (K_b values)for the corresponding nonradioactive compounds, Bu, COOH, and SO₃H, were 0.82, 1.17, and 1.18 × 10⁶ L/mol,respectively. In biodistribution studies, [¹²⁵I]COOH showedhigher radioactivity in the blood and tumors than [¹²⁵I]Bu,whereas [¹²⁵I]SO₃H showed lower radioactivitylevels. Notably, these differences did not correlate with their K_b values. Fluorescence analyses of the compoundsincubated in serum-containing medium demonstrated that the fluorescenceof COOH and Bu plateaued after approximately 1 and 2 h, respectively,whereas that of SO₃H continued to change beyond 3 h, consistentwith a slower time-dependent change in the protein-associated stateof SO₃H. Moreover, longer preincubation times of the radiolabeledasymmetric cyanine dyes in serum-containing medium decreased theirtransfer from the medium to the cellular side, with the greatest decreaseobserved for [¹²⁵I]COOH. These results suggest that functionalgroups affect albumin-binding affinity, serum protein-associated states,and subsequent transfer to the cellular side, thereby influencingblood retention and tumor accumulation. Our findings provide a basisfor the rational optimization of asymmetric cyanine dyes by modulatingalbumin interactions that influence their in vivo behavior.

In our effort todesign an effective ultralow-dose attenuated wholesporozoite (SPZ) vaccine, we explored the use of chemical adjuvantsto increase the immunogenicity of sporozoites. Critical to the potencyof attenuated sporozoites is their ability to migrate to and infectthe liver. We thus used an in vivo mouse challengemodel to compare the effects of external and internal chemical modificationsto sporozoites. We loaded sporozoites with CL307 using two strategies:a supramolecular surface modification adjuvant system (SPZ-SAS(CL307))and a mitochondrial membrane potential system (SPZ-MMP(CL307)). SPZ-SAS(CL307)contained 200 times more CL307 as compared to SPZ-MMP(CL307). However,SPZ-SAS(CL307) could not establish a liver infection after intravenousinjection (25 000 sporozoites), suggesting loss of functionof the sporozoites. On the contrary, SPZ-MMP(CL307) could establishan equivalent liver infection to the control already at a dose of5000 sporozoites. When the immunized mice were challenged, SPZ-MMP(CL307)equaled the protective efficacy of control sporozoites. Our findingsindicate that the MMP system can be used to chemically augment livecells, such as sporozoites, with adjuvants without negatively affectingtheir infectivity and protective efficacy in vivo. For malaria, further testing of different types of adjuvants atdifferent concentrations is needed to ultimately develop vaccineswith increased potency. However, in essence, this platform providesa proof-of-concept for a Trojan horse adjuvanting or drug loadingconcept for any whole-cell vaccine or therapy, within the infectiousdiseases space or beyond in oncological applications.