Production improvement of FK506 in Streptomyces tsukubaensis by metabolic engineering strategy.

AIMS: Study of the effect of isoleucine on the biosynthesis of FK506 and modification of its producing strain to improve the production of FK506. METHODS AND RESULTS: Metabolomics analysis was conducted to explore key changes in the metabolic processes of Streptomyces tsukubaensis Δ68 in medium with and without isoleucine. In-depth analysis revealed that the shikimate pathway, methylmalonyl-CoA, and pyruvate might be the rate-limiting factors in FK506 biosynthesis. Overexpression of involved gene PCCB1 in S. tsukubaensis Δ68, a high-yielding strain Δ68-PCCB1 was generated. Additionally, the amino acids supplement was further optimized to improve FK506 biosynthesis. Finally, FK506 production was increased to 929.6 mg L-1, which was 56.6% higher than that in the starter strain, when supplemented isoleucine and valine at 9 and 4 g L-1, respectively. CONCLUSIONS: Methylmalonyl-CoA might be the key rate-limiting factors in FK506 biosynthesis and overexpression of the gene PCCB1 and further addition of isoleucine and valine could increase the yield of FK506 by 56.6%.

Efficiency Encapsulation of FK506 with New Dual Self-Assembly Multi-Hydrophobic-Core Nanoparticles for Preventing Keratoplasty Rejection.

Nanoparticles self-assembled by amphiphilic copolymers for loading hydrophobic molecules are intensively investigated. However, their hydrophobic molecule-loading capacity is low due to the limitation of hydrophobic groups in these copolymers. In this regard, new lysine oligomer-based multi-hydrophobic side chain polymers (MHCPs) are synthesized by polymerization of γ-benzyl-l glutamate N-carboxy anhydride initiated by side-chain primary amino groups in lysine oligomer. Each hydrophobic side chain in MHCPs can be self-assembled by hydrophobic interaction to form multi-hydrophobic-core nanoparticles (MHC-NPs) with silkworm cocoon-, grape cluster-, and butterfly-like shapes (depending on hydrophobic-side-chains lengths). To increase their stability, MHC-NPs are dually self-assembled with polyethylene glycol-polyglutamic acid through charge interaction. Each hydrophobic core in MHC-NPs serves as a carrier for hydrophobic molecules, endowing their nanostructure with high loading capacity. MHC-NPs are employed to load tacrolimus (also known as FK506), and the loading amount is 18% and the loading efficiency is 80%, which are higher than those of previously reported nanomicelles self-assembled by linear amphiphilic copolymers. Topical administration of FK506-loaded nanoparticle (FK506-NP) can significantly prolong retention of FK506 on the eye surface. FK506-NP exhibits higher in vivo immunosuppressive effects than free FK506 and commercial FK506 eye drop, as well as a better protective effect against immunotoxicity in the corneal grafts after keratoplasty.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.

Tacrolimus nanocrystals microneedle patch for plaque psoriasis.

Plaque psoriasis is characterized by an abnormal thickening of the epidermis, which causes great difficulties for traditional topical drug delivery. Microneedles can pierce the thickened epidermis and deliver drugs to the skin for psoriasis treatment. Tacrolimus is a poorly water-soluble immunosuppressant used for the treatment of psoriasis. In this study, tacrolimus (TAC) nanocrystals (NCs) were produced using a bottom-up technique that dispersed TAC into a sodium hyaluronate-based microneedle patch (MNP), and its therapeutic efficacy was evaluated. The average particle size of the TAC NCs was 259.6 ± 2.3 nm. The mechanical strength of the microneedles was 0.41 ± 0.06 N/needle, which was sufficient to penetrate psoriatic skin. Microneedles were detached from the substrate 10 min after insertion into the psoriasis skin with an insertion depth of 258.8 ± 14.4 µm. The intradermal retention of the MNP (8.40 ± 0.33 µg/cm2) was six times that of the commercial ointment (1.40 ± 0.12 µg/cm2). In pharmacodynamic experiments, results indicated improvement in the phenotypic and histopathological features and reduction in the level of TNF-α, IL-17A, and IL-23 of psoriatic skin treated with TAC NCs MNP. Therefore, MNP loaded with TAC NCs may be a promising approach for psoriasis treatment.

Structural insights into Plasmodium PPIases.

Malaria is one of the most prevalent infectious diseases posing a serious challenge over the years, mainly owing to the emergence of drug-resistant strains, sparking a need to explore and identify novel protein targets. It is a well-known practice to adopt a chemo-genomics approach towards identifying targets for known drugs, which can unravel a novel mechanism of action to aid in better drug targeting proficiency. Immunosuppressive drugs cyclosporin A, FK506 and rapamycin, were demonstrated to inhibit the growth of the malarial parasite, Plasmodium falciparum. Peptidyl prolyl cis/trans isomerases (PPIases), comprising cylcophilins and FK506-binding proteins (FKBPs), the specific target of these drugs, were identified in the Plasmodium parasite and proposed as an antimalarial drug target. We previously attempted to decipher the structure of these proteins and target them with non-immunosuppressive drugs, predominantly on FKBP35. This review summarizes the structural insights on Plasmodium PPIases, their inhibitor complexes and perspectives on drug discovery.

Distribution evaluation of tacrolimus in the ascitic fluid of liver transplant recipients with liver cirrhosis by a sensitive ultra-performance liquid chromatography-tandem mass spectrometry method.

Tacrolimus has a narrow therapeutic index and large individual differences in pharmacokinetics. The distribution of tacrolimus in ascitic fluid and its influence on whole-blood tacrolimus were unclear. In this study, a sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was established and validated for the quantification of tacrolimus in the ascitic fluid of liver transplant recipients. Chromatographic separation was achieved on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column (2.1 × 100 mm, 3.5 µm). Mass spectrometry was performed in multiple reaction monitoring conditions of transitions m/z 821.4→768.5 for tacrolimus. The concentrations of tacrolimus in the ascitic fluid range from 0.2 to 3.0 ng/mL, accounting for 1.19-31.87% of whole-blood tacrolimus concentrations. A linear mixed model showed a statistically significant positive correlation between the steady-state trough blood concentration of tacrolimus and the corresponding amount of tacrolimus excreted in the ascitic fluid for 24 consecutive hours, especially after normalization by daily dose per unit body weight. These data suggested that the distribution of tacrolimus in the ascitic fluid has great individual differences. The whole-blood tacrolimus concentration, dose per unit body weight, and other confounding factors may contribute to the excretion of tacrolimus in ascitic fluid, but the influence of tacrolimus excretion in drained ascitic fluid on the whole-blood tacrolimus concentration is negligible.

Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs.

Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

Rigorous analysis of the interaction between proteins and low water-solubility drugs by qNMR-aided NMR titration experiments.

Drugs are designed and validated based on physicochemical data on their interactions with target proteins. For low water-solubility drugs, however, quantitative analysis is practically impossible without accurate estimation of precipitation. Here we combined quantitative NMR with NMR titration experiments to rigorously quantify the interaction of the low water-solubility drug pimecrolimus with its target protein FKBP12. Notably, the dissociation constants estimated with and without consideration of precipitation differed by more than tenfold. Moreover, the method enabled us to quantitate the FKBP12-pimecrolimus interaction even under a crowded condition established using the protein crowder BSA. Notably, the FKBP12-pimecrolimus interaction was slightly hampered under the crowded environment, which is explained by transient association of BSA with the drug molecules. Collectively, the described method will contribute to both quantifying the binding properties of low water-solubility drugs and to elucidating the drug behavior in complex crowded solutions including living cells.

Tacrolimus-loaded chitosan nanoparticles for enhanced skin deposition and management of plaque psoriasis.

Tacrolimus is a natural macrolide that exhibits an anti-proliferative action by T-lymphocytic cells inhibition. Hence, it was tested as a potential topical treatment to improve and control psoriatic plaques. In this study, for the first time the lipophilic tacrolimus in chitosan nanoparticles was used to achieve the desired response and dermal retention of the drug using a modified ionic gelation technique. The hydrophobic drug, tacrolimus, was successfully encapsulated into the synthesized positively-charged particles (140.8 nm ± 50.0) and EE of (65.5% ± 1.3). Local skin deposition of the drug was significantly enhanced with 82.0% ± 0.6 of the drug retained in the skin compared to 34.0% ± 0.9 from tarolimus® ointment. An outstanding response to the prepared formula was the enhanced hair growth rate in the treated animals, which can be considered an excellent sign of the skin recovery from the induced psoriatic plaques after only three days of treatment.

Macrocyclic FKBP51 Ligands Define a Transient Binding Mode with Enhanced Selectivity.

Subtype selectivity represents a challenge in many drug discovery campaigns. A typical example is the FK506 binding protein 51 (FKBP51), which has emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs. FKBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During a macrocyclization pilot study, we observed that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with a new binding mode featuring a transient conformation, which is disfavored for the small FKBPs. Using a conformation-sensitive assay we show that this binding mode occurs in solution and is characteristic for this new class of compounds. The discovered macrocycles are non-immunosuppressive, engage FKBP51 in cells, and block the cellular effect of FKBP51 on IKKα. Our findings provide a new chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

Combined local delivery of tacrolimus and stem cells in hydrogel for enhancing peripheral nerve regeneration.

The application of scaffold-based stem cell transplantation to enhance peripheral nerve regeneration has great potential. Recently, the neuroregenerative potential of tacrolimus (a U.S. Food and Drug Administration-approved immunosuppressant) has been explored. In this study, a fibrin gel-based drug delivery system for sustained and localized tacrolimus release was combined with rat adipose-derived mesenchymal stem cells (MSC) to investigate cell viability in vitro. Tacrolimus was encapsulated in poly(lactic-co-glycolic) acid (PLGA) microspheres and suspended in fibrin hydrogel, using concentrations of 0.01 and 100 ng/ml. Drug release over time was measured. MSCs were cultured in drug-released media collected at various days to mimic systemic exposure. MSCs were combined with (i) hydrogel only, (ii) empty PLGA microspheres in the hydrogel, (iii) 0.01, and (iv) 100 ng/ml of tacrolimus PLGA microspheres in the hydrogel. Stem cell presence and viability were evaluated. A sustained release of 100 ng/ml tacrolimus microspheres was observed for up to 35 days. Stem cell presence was confirmed and cell viability was observed up to 7 days, with no significant differences between groups. This study suggests that combined delivery of 100 ng/ml tacrolimus and MSCs in fibrin hydrogel does not result in cytotoxic effects and could be used to enhance peripheral nerve regeneration.

Phenotypic screen identifies calcineurin-sparing FK506 analogs as BMP potentiators for treatment of acute kidney injury.

Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.

Anaphylaxis Induced by Intravenous Tacrolimus Administration During Kidney Transplant Surgery: A Case Report.

A 35-year-old male patient with end-stage renal disease due to vesicoureteral reflux preemptively received a renal graft from his father. The patient had a history of allergy to contrast-enhancing media. He received oral tacrolimus (TAC) and mycophenolate mofetil without any problems for 2 days before kidney transplantation. During the induction period of the surgery, his systolic blood pressure (sBP) decreased to 60 mmHg approximately 1 hour after initiating intravenous tacrolimus (TAC-IV) and intravenous piperacillin (PIPC), and the anesthesiologist suspected drug-induced anaphylaxis and stopped administration of the medications. Because TAC had been administered preoperatively without any adverse events, PIPC was suspected as the causative agent of the anaphylaxis. After the patient's hemodynamics returned to baseline, TAC-IV was restarted. However, his sBP rapidly decreased to 40 mmHg and the patient developed wheezing. He was diagnosed with drug-induced anaphylaxis due to castor oil derivatives in the TAC-IV formulation. The patient's sBP was restored with the administration of some vasopressors, and kidney transplantation was then performed without difficulty. Two days after kidney transplantation, oral TAC was administered without anaphylaxis. Clinicians should consider that not only the drug itself but also its additives or metabolites could induce anaphylaxis.

Preparation and Characterization of Tacrolimus-Loaded SLNs in situ Gel for Ocular Drug Delivery for the Treatment of Immune Conjunctivitis.

BACKGROUND: The aim of this study is to develop a novel in situ gel of tacrolimus-loaded SLNs (solid lipid nanoparticles) for ocular drug delivery. METHODS: The optimal formulation was characterized by surface morphology, particle size, zeta potential, entrapment efficiency, drug loading and in vitro release behavior. In vivo studies were also conducted to evaluate the pharmacokinetic and pharmacodynamic results. RESULTS: In this study, TAC-SLNs ISG were prepared using homogenization followed by probe sonication method. The average particle size of TAC-SLNs ISG was observed to be 122.3±4.3 nm. Compared with TAC-SLNs, in situ gel did not increase particle size, and there was no significant difference between them. The results of viscosity measurement showed that TAC SLNs-ISG were typical of pseudo plastic systems and showed a marked increase in viscosity as temperature increased and ultimately formed a rigid gel (32°C). In vitro and in vivo studies illustrated the sustained release model of the drug from TAC-SLNs ISG. Animal model showed that TAC-SLNs ISG had good pharmacodynamics when compared with eye drops and SLNs. CONCLUSION: Our results demonstrated that TAC SLNs-ISG had the potential for being an ideal ocular drug delivery system.

The Rise of Molecular Glues.

2021 marks the 30th anniversary of the revelation that cyclosporin A and FK506 act in a way previously not seen-as "molecular glues" that induce neo-protein-protein associations. As a torrent of new molecular-glue probes and medicines are fueling interest in this field, I explore the arc of this story.

Volumetric absorptive microsampling for the quantification of tacrolimus in capillary blood by high performance liquid chromatography-tandem mass spectrometry.

Volumetric absorptive microsampling (VAMS) is an innovative alternative strategy to venipuncture for monitoring tacrolimus levels in transplant recipients. In this study, we aimed to validate a new high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying tacrolimus in blood collected by VAMS. Tacrolimus was extracted from dried blood tips in an original process involving sonication, protein precipitation and salting out. The assay was validated in accordance with EMA and IATDMCT guidelines. For clinical validation, the tacrolimus concentrations measured in liquid venous whole blood (with the reference method) were compared with those measured in capillary whole blood collected simultaneously with VAMS by a nurse. The assay was then used to monitor tacrolimus exposure in transplant recipients. The method was linear, sensitive and fast. Within-day and between-day precisions and overall bias were within ±15%. No significant hematocrit effect was observed. The matrix effect was negligible and recovery exceeded 80% for every concentration and hematocrit levels. Tacrolimus was stable in blood collected by VAMS for 1 week at room temperature, 48 h at 60 °C and 4 °C and 1 month at -80 °C. Clinical validation (n = 42 paired samples) demonstrated a strong correlation between the two methods (r = 0.97 Pearson correlation). Bland-Altman analysis revealed that more than 90% of the differences between VAMS and liquid blood paired concentrations were within the ±20% acceptable range. The method had a satisfactory analytical performance and fulfilled clinical requirements. This minimally invasive VAMS-based assay appears reliable for the determination of tacrolimus levels in blood from transplanted patients.

Surface functionalized mesoporous silica nanoparticles for intravitreal application of tacrolimus.

Tacrolimus (TAC), a potent immunosuppressive macrolide, has been investigated for ocular diseases due to promising results in the treatment of anterior and posterior segments eye diseases. Mesoporous and functionalized silica nanoparticles show potential as TAC delivery platforms owing to their interesting characteristic as large surface area, uniform pore size distribution, high pore volume, and excellent biocompatibility. The purpose of this study was to incorporate TAC in functionalized silica nanoparticles with 3-aminopropyltriethoxysilane (MSNAPTES) and investigate the safety and biocompatibility of the systems. The MSNAPTES and MSNAPTES TAC nanoparticles were characterized. The in vitro cytotoxicity of MSNAPTES and MSNAPTES load with TAC (MSNAPTES-TAC) in retinal pigment epithelial cells (ARPE-19) was determined, chorioallantoic membrane (CAM) assay model was used to investigate the in vivo biocompatibility, and safety of intravitreal injection was evaluated using clinical examination (assessment of intraocular pressure and indirect fundus ophthalmoscopy), electroretinographic (ERG) and histologic studies in rats' eyes. The elemental analysis (CHN), thermogravimetric (TGA), photon correlation spectroscopy and Fourier transform infrared (FTIR) analysis confirmed the presence of functionalized agent and TAC in the MSNAPTES nanoparticles. TAC loading was estimated at 7% for the MSNAPTES TAC nanoparticles. MSNAPTES and MSNAPTES TAC did not present in vitro cytotoxicity. The drug delivery systems showed good biocompatibility on CAM. No retinal abnormalities, vitreous hemorrhage, neovascularization, retinal detachment, and optic nerve atrophy were observed during the in vivo study. Follow-up ERGs showed no changes in the function of the retina cells after 15 days of intravitreal injection, and histopathologic observations support these findings. In conclusion, MSNAPTES TAC was successfully synthesized, and physicochemical analyses confirmed the presence of TAC in the nanoparticles. In vitro and in vivo studies indicated that MSNAPTES TAC was safe to intravitreal administration. Taking into account the enormous potential of MSNAPTES to carry TAC, this platform could be a promising strategy for TAC ocular drug delivery in the treatment of eye diseases.

Comparison of the Impact of Pharmacogenetic Variability on the PK of Slow Release and Immediate Release Tacrolimus Formulations.

Tacrolimus-modified release formulations allow for once-daily dosing, and adherence is better compared to the twice-daily immediate release formulation. When patients are switched from one formulation to another, variable changes in drug concentrations are observed. Current data suggest that the changes in drug exposure are larger in patients who express the CYP3A5 enzyme (CYP3A5 *1/*3 or *1/*1) compared to nonexpressers (CYP3A5*3/*3). Possibly, these differences are due to the fact that in the upper region of the small intestine CYP3A activity is higher, and that this expression of CYP3A decreases towards the more distal parts of the gut. Modified release formulations may therefore be subject to a less presystemic metabolism. However, the full implications of pharmacogenetic variants affecting the expression and function of drug transporters in the gut wall and of enzymes involved in phase I and phase II metabolism on the different formulations are incompletely understood, and additional studies are required. Conclusions: In all patients in whom the formulation of tacrolimus is changed, drug levels need to be checked to avoid clinically relevant under- or overexposure. In patients with the CYP3A5 expresser genotype, this recommendation is even more important, as changes in drug exposure can be expected.

Using thin film freezing to minimize excipients in inhalable tacrolimus dry powder formulations.

We investigated the feasibility of preparing high-potency tacrolimus dry powder for inhalation using thin film freezing (TFF). We found that using ultra-rapid freezing can increase drug loading up to 95% while maintaining good aerosol performance. Drug loading affected the specific surface area and moisture sorption of TFF formulations, but it did not affect the chemical stability, physical stability, and dissolution of tacrolimus. Tacrolimus remained amorphous after storage at 40 °C/75% RH, and 25 °C/60% RH for up to 6 months. Lactose functioned as a bulking agent, and it had little to no effect as a stabilizer for amorphous tacrolimus due to a lack of interaction between the drug and excipient. Additionally, the aerosol performance of TFF tacrolimus/lactose (95/5) did not significantly change after six months of storage at 25 °C/60% RH. For processing parameters, the solids content and the processing temperature did not affect the aerosol performance of tacrolimus. Furthermore, both low- and high-resistance RS01 showed optimal and consistent aerosol performance over the 1-4 kPa pressure drop range. In conclusion, TFF is a suitable technology for producing inhalable powder that contain high drug loading and have less flow rate dependence.

Carboxymethyl Cellulose-Coated Tacrolimus Nonspherical Microcrystals for Improved Therapeutic Efficacy of Dry Eye.

Dry eye (DE) is a highly prevalent ocular surface disease which affects the quality of life and results in low working efficiency. Frequent instillation is required due to low bioavailability of conventional eye drops. The aim of this study is to develop a novel formulation of tacrolimus (TAC), routinely prescribed for DE, by combination of the microcrystal technology and layer-by-layer assembly. First, nonspherical tacrolimus microcrystals (TAC MCs) are synthesized by antisolvent-induced precipitation. These TAC MCs are modified by alternate deposition of poly(allylamine hydrochloride) (PAH) and carboxymethyl cellulose (CMC) subsequently to obtain CMC-coated TAC MCs (TAC-(PAH/CMC)3 ). The resultant formulations are evaluated in vivo in a mouse DE model induced by an intelligently controlled environmental system. Compared with commercially available TAC eye drops and the TAC MCs counterpart, TAC-(PAH/CMC)3 exhibits superior therapeutic performance with reduced drug instillation frequency, which is attributed to the nonspherical geometry of MCs, the lubricant, mucoadhesive effect of CMC, and the anti-inflammatory function of TAC. Therefore, TAC-(PAH/CMC)3 represents a better option for the management of DE.