Novel bispecific nanobody mitigates experimental intestinal inflammation in mice by targeting TNF-α and IL-23p19 bioactivities.

BACKGROUND: Inflammatory bowel diseases (IBDs) pose significant challenges in terms of treatment non-response, necessitating the development of novel therapeutic approaches. Although biological medicines that target TNF-α (tumour necrosis factor-α) have shown clinical success in some IBD patients, a substantial proportion still fails to respond. METHODS: We designed bispecific nanobodies (BsNbs) with the ability to simultaneously target human macrophage-expressed membrane TNF-α (hmTNF-α) and IL-23. Additionally, we fused the constant region of human IgG1 Fc (hIgG1 Fc) to BsNb to create BsNb-Fc.  Our study encompassed in vitro and in vivo characterization of BsNb and BsNb-Fc. RESULTS: BsNb-Fc exhibited an improved serum half-life, targeting capability and effector function than BsNb. It's demonstrated that BsNb-Fc exhibited superior anti-inflammatory effects compared to the anti-TNF-α mAb (infliximab, IFX) combined with anti-IL-12/IL-23p40 mAb (ustekinumab, UST) by Transwell co-culture assays. Notably, in murine models of acute colitis brought on by 2,4,6-trinitrobenzene sulfonic acid（TNBS) and dextran sulphate sodium (DSS), BsNb-Fc effectively alleviated colitis severity. Additionally, BsNb-Fc outperformed the IFX&UST combination in TNBS-induced colitis, significantly reducing colon inflammation in mice with colitis produced by TNBS and DSS. CONCLUSION: These findings highlight an enhanced efficacy and improved biostability of BsNb-Fc, suggesting its potential as a promising therapeutic option for IBD patients with insufficient response to TNF-α inhibition. KEY POINTS: A bispecific nanobody (BsNb) was created to target TNF-α and IL-23p19, exhibiting high affinity and remarkable stability. BsNb-Fc inhibited the release of cytokines in CD4+T cells during co-culture experiments. BsNb-Fc effectively alleviated colitis severity in mouse model with acute colitis induced by DSS or TNBS, outperforming the IFX&UST combination.

Diallyl disulfide induces DNA damage and growth inhibition in colorectal cancer cells by promoting POU2F1 ubiquitination.

Previous studies have demonstrated that diallyl disulfide (DADS) exhibits potent anti-tumor activity. However, the pharmacological actions of DADS in inhibiting the growth of colorectal cancer (CRC) cells have not been clarified. Herein, we show that DADS treatment impairs the activation of the pentose phosphate pathway (PPP) to decrease PRPP (5-phosphate ribose-1-pyrophosphate) production, enhancing DNA damage and cell apoptosis, and inhibiting the growth of CRC cells. Mechanistically, DADS treatment promoted POU2F1 K48-linked ubiquitination and degradation by attenuating the PI3K/AKT signaling to up-regulate TRIM21 expression in CRC cells. Evidently, TRIM21 interacted with POU2F1, and induced the K272 ubiquitination of POU2F1. The effects of DADS on the enhanced K272 ubiquitination of POU2F1, the PPP flux, PRPP production, DNA damage and cell apoptosis as well as the growth of CRC tumors in vivo were significantly mitigated by TRIM21 silencing or activating the PI3K signaling in CRC cells. Conversely, the effects of DADS were enhanced by TRIM21 over-expression or inhibiting the PI3K/AKT signaling in CRC cells. Collectively, our findings reveal a novel mechanism by which DADS suppresses the growth of CRC by promoting POU2F1 ubiquitination, and may aid in design of novel therapeutic intervention of CRC.

The roles and molecular mechanisms of non-coding RNA in cancer metabolic reprogramming.

One of the key features of cancer is energy metabolic reprogramming which is tightly related to cancer proliferation, invasion, metastasis, and chemotherapy resistance. NcRNAs are a class of RNAs having no protein-coding potential and mainly include microRNAs, lncRNAs and circRNAs. Accumulated evidence has suggested that ncRNAs play an essential role in regulating cancer metabolic reprogramming, and the altered metabolic networks mediated by ncRNAs primarily drive carcinogenesis by regulating the expression of metabolic enzymes and transporter proteins. Importantly, accumulated research has revealed that dysregulated ncRNAs mediate metabolic reprogramming contributing to the generation of therapeutic tolerance. Elucidating the molecular mechanism of ncRNAs in cancer metabolic reprogramming can provide promising metabolism-related therapeutic targets for treatment as well as overcome therapeutic tolerance. In conclusion, this review updates the latest molecular mechanisms of ncRNAs related to cancer metabolic reprogramming.

Ferritinophagy: research advance and clinical significance in cancers.

Ferritinophagy, a process involving selective autophagy of ferritin facilitated by nuclear receptor coactivator 4 (NCOA4), entails the recognition of ferritin by NCOA4 and subsequent delivery to the autophagosome. Within the autophagosome, ferritin undergoes degradation, leading to the release of iron in the lysosome. It is worth noting that excessive iron levels can trigger cell death. Recent evidence has elucidated the significant roles played by ferritinophagy and ferroptosis in regulation the initiation and progression of cancer. Given the crucial role of ferritinophagy in tumor biology, it may serve as a potential target for future anti-tumor therapeutic interventions. In this study, we have provided the distinctive features of ferritinophagy and its distinctions from ferroptosis. Moreover, we have briefly examined the fundamental regulatory mechanisms of ferritinophagy, encompassing the involvement of the specific receptor NCOA4, the Nrf2/HO-1 signaling and other pathways. Subsequently, we have synthesized the current understanding of the impact of ferritinophagy on cancer progression and its potential therapeutic applications, with a particular emphasis on the utilization of chemotherapy, nanomaterials, and immunotherapy to target the ferritinophagy pathway for anti-tumor purposes.

Affinity maturation of antibody fragments: A review encompassing the development from random approaches to computational rational optimization.

Routinely screened antibody fragments usually require further in vitro maturation to achieve the desired biophysical properties. Blind in vitro strategies can produce improved ligands by introducing random mutations into the original sequences and selecting the resulting clones under more and more stringent conditions. Rational approaches exploit an alternative perspective that aims first at identifying the specific residues potentially involved in the control of biophysical mechanisms, such as affinity or stability, and then to evaluate what mutations could improve those characteristics. The understanding of the antigen-antibody interactions is instrumental to develop this process the reliability of which, consequently, strongly depends on the quality and completeness of the structural information. Recently, methods based on deep learning approaches critically improved the speed and accuracy of model building and are promising tools for accelerating the docking step. Here, we review the features of the available bioinformatic instruments and analyze the reports illustrating the result obtained with their application to optimize antibody fragments, and nanobodies in particular. Finally, the emerging trends and open questions are summarized.

Protective Immunity against Chlamydia psittaci Lung Infection Induced by a DNA Plasmid Vaccine Carrying CPSIT_p7 Gene Inhibits Dissemination in BALB/c Mice.

Chlamydia psittaci (C. psittaci), a zoonotic pathogen, poses a potential threat to public health security and the development of animal husbandry. Vaccine-based preventative measures for infectious diseases have a promising landscape. DNA vaccines, with many advantages, have become one of the dominant candidate strategies in preventing and controlling the chlamydial infection. Our previous study showed that CPSIT_p7 protein is an effective candidate for a vaccine against C. psittaci. Thus, this study evaluated the protective immunity of pcDNA3.1(+)/CPSIT_p7 against C. psittaci infection in BALB/c mice. We found that pcDNA3.1(+)/CPSIT_p7 can induce strong humoral and cellular immune responses. The IFN-γ and IL-6 levels in the infected lungs of mice immunized with pcDNA3.1(+)/CPSIT_p7 reduced substantially. In addition, the pcDNA3.1(+)/CPSIT_p7 vaccine diminished pulmonary pathological lesions and reduced the C. psittaci load in the lungs of infected mice. It is worth noting that pcDNA3.1(+)/CPSIT_p7 suppressed C. psittaci dissemination in BALB/c mice. In a word, these results demonstrate that the pcDNA3.1(+)/CPSIT_p7 DNA vaccine has good immunogenicity and immunity protection effectiveness against C. psittaci infection in BALB/c mice, especially pulmonary infection, and provides essential practical experience and insights for the development of a DNA vaccine against chlamydial infection.

Design of nanobody-based bispecific constructs by in silico affinity maturation and umbrella sampling simulations.

Random mutagenesis is the natural opportunity for proteins to evolve and biotechnologically it has been exploited to create diversity and identify variants with improved characteristics in the mutant pools. Rational mutagenesis based on biophysical assumptions and supported by computational power has been proposed as a faster and more predictable strategy to reach the same aim. In this work we confirm that substantial improvements in terms of both affinity and stability of nanobodies can be obtained by using combinations of algorithms, even for binders with already high affinity and elevated thermal stability. Furthermore, in silico approaches allowed the development of an optimized bispecific construct able to bind simultaneously the two clinically relevant antigens TNF-α and IL-23 and, by means of its enhanced avidity, to inhibit effectively the apoptosis of TNF-α-sensitive L929 cells. The results revealed that salt bridges, hydrogen bonds, aromatic-aromatic and cation-pi interactions had a critical role in increasing affinity. We provided a platform for the construction of high-affinity bispecific constructs based on nanobodies that can have relevant applications for the control of all those biological mechanisms in which more than a single antigen must be targeted to increase the treatment effectiveness and avoid resistance mechanisms.

Breaking through the therapeutic ceiling of inflammatory bowel disease: Dual-targeted therapies.

Emerging biologics and small-molecule drugs have changed the clinical status quo of inflammatory bowel disease (IBD). However, current treatments remain at a standstill in terms of response and remission in many cases. Accumulating evidence indicates that dual-targeted therapy (DTT) could be promising in overcoming the existing ceiling of IBD treatment. However, data on the efficacy and safety of DTT on Crohn's disease and ulcerative colitis are still limited or insufficient. Moreover, there is a lack of studies delineating the mechanisms of DTT. Given that various targeted drugs have different targets among the extensive redundant inflammatory networks, DTT could result in various outcomes. In this review, we have summarized the current data on the safety, effectiveness, and clinical development status of novel targeted drugs related to refractory IBD, and have explored the mechanism of action of therapy. We have categorized therapeutic agents into "Therapeutic Agents Targeting Cellular Signaling Pathways" and "Therapeutic Agents Targeting Leukocyte Trafficking" based on the different therapeutic targets, and also by classifying therapeutic agents targeting the cellular signaling pathways into "JAK-dependent" and "JAK-independent," and placed the existing drug combinations into 3 categories based on their mechanisms, namely, overlapping, synergistic, and complementary effects. Lastly, we have proposed the possible mechanisms of DTT to conceive a theoretical framework for clinical decision-making and further drug development and research from an IBD standpoint.

A Biologically Inspired Model of Collective Bio-Nanomachine Rotation via Chemical and Physical Interactions.

Creating a large-scale functional system from a group of bio-nanomachines is a key objective for engineering innovative applications. This paper aims to create a large-scale system of bio-nanomachines and proposes a collective rotational motion model to describe their behaviour. The proposed model is based on the notion that spinning objects are stable against perturbations, meaning that a rotating cluster of bio-nanomachines may be stable and suitable for large-scale bio-nanomachine systems to be engineered for applications in dynamic and noisy environments. In developing the model, we draw inspiration from biological pattern formation, where biological entities interact chemically and physically to form a functional structure. Accordingly, we develop a collective rotational motion model of bio-nanomachines based on their chemical and physical interactions. Through modeling and simulations, we gain insight into the design and engineering of rotating clusters of bio-nanomachines. This paper demonstrates the importance of physical interactions in creating system-level functionality from a group of bio-nanomachines, giving rise to new challenges and opportunities in molecular communication research.

ATP levels influence cell movement during the mound phase in Dictyostelium discoideum as revealed by ATP visualization and simulation.

Cell migration plays an important role in multicellular organism development. The cellular slime mold Dictyostelium discoideum is a useful model organism for the study of cell migration during development. Although cellular ATP levels are known to determine cell fate during development, the underlying mechanism remains unclear. Here, we report that ATP-rich cells efficiently move to the central tip region of the mound against rotational movement during the mound phase. A simulation analysis based on an agent-based model reproduces the movement of ATP-rich cells observed in the experiments. These findings indicate that ATP-rich cells have the ability to move against the bulk flow of cells, suggesting a mechanism by which high ATP levels determine the cell fate of differentiation.

Modeling the effect of age on quantiles of the incubation period distribution of COVID-19.

BACKGROUND: The novel coronavirus SARS-CoV-2 (coronavirus disease 2019, COVID-19) has caused serious consequences on many aspects of social life throughout the world since the first case of pneumonia with unknown etiology was identified in Wuhan, Hubei province in China in December 2019. Note that the incubation period distribution is key to the prevention and control efforts of COVID-19. This study aimed to investigate the conditional distribution of the incubation period of COVID-19 given the age of infected cases and estimate its corresponding quantiles from the information of 2172 confirmed cases from 29 provinces outside Hubei in China. METHODS: We collected data on the infection dates, onset dates, and ages of the confirmed cases through February 16th, 2020. All the data were downloaded from the official websites of the health commission. As the epidemic was still ongoing at the time we collected data, the observations subject to biased sampling. To address this issue, we developed a new maximum likelihood method, which enables us to comprehensively study the effect of age on the incubation period. RESULTS: Based on the collected data, we found that the conditional quantiles of the incubation period distribution of COVID-19 vary by age. In detail, the high conditional quantiles of people in the middle age group are shorter than those of others while the low quantiles did not show the same differences. We estimated that the 0.95-th quantile related to people in the age group 23 ∼55 is less than 15 days. CONCLUSIONS: Observing that the conditional quantiles vary across age, we may take more precise measures for people of different ages. For example, we may consider carrying out an age-dependent quarantine duration in practice, rather than a uniform 14-days quarantine period. Remarkably, we may need to extend the current quarantine duration for people aged 0 ∼22 and over 55 because the related 0.95-th quantiles are much greater than 14 days.

Activity Components from Gynostemma pentaphyllum for Preventing Hepatic Fibrosis and of Its Molecular Targets by Network Pharmacology Approach.

Hepatic fibrosis would develop into cirrhosis or cancer without treating. Hence, it is necessary to study the mechanism and prevention methods for hepatic fibrosis. Gynostemma pentaphyllum is a traditional medicinal material with a high medicinal and health value. In this study, nineteen compounds obtained from G. pentaphyllum were qualitative and quantitative by HPLC-FT-ICR MS and HPLC-UV, respectively. Among them, the total content of 19 gypenosides accurately quantified reaches 72.21 mg/g and their anti-proliferation against t-HSC/Cl-6 cells indicated compound 19 performed better activity (IC50: 28.1 ± 2.0 µM) than the other compounds. Further network pharmacology study demonstrated that compound 19 mainly plays an anti-fibrosis role by regulating the EGFR signaling pathway, and the PI3K-Akt signaling pathway. Overall, the verification result indicated that compound 19 appeared to be nontoxic to LO2, was able to modulate the PI3K/Akt signal, led to subG1 cells cycle arrest and the activation of mitochondrial-mediated apoptosis of t-HSC/Cl-6 cells for anti-hepatic fibrosis.

Hair Growth Promoting Activity of Cedrol Nanoemulsion in C57BL/6 Mice and Its Bioavailability.

As the main component of Platycladus orientalis, cedrol has known germinal activity. A range of cedrol formulations have been developed to prevent hair-loss, but compliance remains key issues. In this study, we prepared cedrol nanoemulsion (CE-NE) and determined the particle size and PDI (polydispersion coefficient), investigated the hair growth activity and studied the bioavailability in vitro and in vivo. Results showed that the average particle size of CE-NE is 14.26 ± 0.16 nm, and the PDI value is 0.086 ± 0.019. In vitro drug release investigation and drug release kinetics analysis showed release profile of CE from nanoparticles demonstrates the preferred partition of CE in buffer pH 4.0, the release profile of CE-NE showed a first-order kinetics reaching around 36.7% after 6 h at 37 °C. We artificially depilated the back hair of C57BL/6 mice and compared the efficacy of a designed cedrol nanoemulsion to an existing ointment group. The hair follicles were imaged and quantified using a digital photomicrograph. The results showed that compared with the ointment, CE-NE had positive effects on hair growth, improved drug solubility. Compared with the ointment and 2% minoxidil groups, 50 mg/mL CE-NE led to more robust hair growth. Pharmacokinetics analysis showed that the AUC0-t of CE-NE was 4-fold higher than that of the ointment group, confirming that the bioavailability of the nanoemulsion was greater than that of the ointment. CE-NE also significantly reduced the hair growth time of model mice and significantly increased the growth rate of hair follicles. In conclusion, these data suggest that the nanoemulsion significantly improved the pharmacokinetic properties and hair growth effects cedrol, enhancing its efficacy in vitro and in vivo.

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo.

OBJECTIVE: We aimed to develop a novel anti-HIF-1α intrabody to decrease gemcitabine resistance in pancreatic cancer patients. METHODS: Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1α VHH212 [a single-domain antibody (nanobody)]. Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α (HIF-1α) and VHH212. The real-time polymerase chain reaction (PCR) and Western blot analyses were performed to identify the expressions of HIF-1α and VEGF-A in pancreatic ductal adenocarcinoma cell lines. The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay. Finally, a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment. Immunohistochemistry analysis was conducted to detect the expressions of HIF-1α and VEGF-A in tumor tissues. RESULTS: VHH212 was stably expressed in tumor cells with low cytotoxicity, high affinity, specific subcellular localization, and neutralization of HIF-1α in the cytoplasm or nucleus. The binding affinity between VHH212 and the HIF-1α PAS-B domain was 42.7 nM. Intrabody competitive inhibition of the HIF-1α heterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro. Compared with single agent gemcitabine, co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44% tumor inhibition. CONCLUSIONS: We developed an anti-HIF-1α nanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer. The combination of VHH212 and gemcitabine significantly inhibited tumor development. These results suggested that combined use of anti-HIF-1α nanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region.

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains.

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment.

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative to conventional monoclonal antibodies also for clinical applications. Furthermore, they do not solely represent a substitute for monoclonal antibodies but their unique features allow expanding the applications of biotherapeutics and changes the pattern of disease treatment. Nanobodies possess the double advantage of being small and simple to engineer. This combination has promoted extremely diversified approaches to design nanobody-based constructs suitable for particular applications. Both the format geometry possibilities and the functionalization strategies have been widely explored to provide macromolecules with better efficacy with respect to single nanobodies or their combination. Nanobody multimers and nanobody-derived reagents were developed to image and contrast several cancer diseases and have shown their effectiveness in animal models. Their capacity to block more independent signaling pathways simultaneously is considered a critical advantage to avoid tumor resistance, whereas the mass of these multimeric compounds still remains significantly smaller than that of an IgG, enabling deeper penetration in solid tumors. When applied to CAR-T cell therapy, nanobodies can effectively improve the specificity by targeting multiple epitopes and consequently reduce the side effects. This represents a great potential in treating malignant lymphomas, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma and solid tumors. Apart from cancer treatment, multispecific drugs and imaging reagents built with nanobody blocks have demonstrated their value also for detecting and tackling neurodegenerative, autoimmune, metabolic, and infectious diseases and as antidotes for toxins. In particular, multi-paratopic nanobody-based constructs have been developed recently as drugs for passive immunization against SARS-CoV-2 with the goal of impairing variant survival due to resistance to antibodies targeting single epitopes. Given the enormous research activity in the field, it can be expected that more and more multimeric nanobody molecules will undergo late clinical trials in the next future. Systematic Review Registration.

In vitro affinity maturation to improve the efficacy of a hypoxia-inducible factor 1α single-domain intrabody.

Affinity is an important property of therapeutic antibodies, so improving affinity is critical to the biological activity and clinical efficacy. An anti-HIF-1α nanobody, VHH212, was screened via a native ribosome display library with a 26.6 nM of KD value was used as the parent. In this paper, a Venn-intersection of multi-algorithms screening (VIMAS) strategy for computer-aided binding affinity prediction was designed. Homology modeling and protein docking methods were used to substitute the need for a crystal structure. Finally, a mutant with a 17.5-fold enhancement in binding affinity (1.52 nM) was obtained by using the VIMAS strategy. Furthermore, the biological activity of mutants was verified at the cellular level. Targeting HIF-1α can sensitize PDAC (pancreatic ductal adenocarcinoma) tumors to gemcitabine, which is a potential co-treatment method for pancreatic cancer patients. Our results showed that the cytotoxicity of gemcitabine on pancreatic cancer cell lines increased with the enhanced-affinity of an intrabody under combined treatment.

Homology Modeling-Based in Silico Affinity Maturation Improves the Affinity of a Nanobody.

Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.

Anti-inflammatory and analgesic activity based on polymorphism of cedrol in mice.

Alternate forms of drug crystals display different physicochemical properties. These include stability, dissolution rate, bioavailability and solubility, which can affect pharmacokinetics and pharmacodynamics. It is therefore important to compare the crystal forms of cedrol to obtain optimal anti-inflammatory and analgesic effects. This study, for the first time, obtained and reports three novel forms (I-III) of cedrol polymorphs. The three forms of cedrol were recrystallized from seven organic solvents by slow cooling or volatilization and identified by thermal analysis, fourier transform infrared spectroscopy, scanning electron microscopy and powder X-ray diffraction analysis. Form I originated from acetone and cyclohexane. Form II was obtained from ethanol, ethyl acetate, acetonitrile and n-hexane. Form III was recrystallized from methanol. The anti-inflammatory and analgesic activities of the three crystalline forms were evaluated by acetic acid induced writhing in mice, the hot plate method, carrageenan induced mouse paw edema models, Xylene-induced mouse ear edema models and cotton pellet-induced mouse granuloma models. Experimental results revealed that the highest performance was achieved from Form I. These findings are of great significance during the early research study of cedrol polymorphs.