Preformulation Studies and Enabling Formulation Selection for an Insoluble Compound at Preclinical Stage-From In Vitro, In Silico to In Vivo.

The objective of this work was to identify an enabling formulation for an insoluble compound ZL006 with potency of boosting leukocytes after chemotherapy. The low oral bioavailability (<1%) of its conventional suspension was the hurdle for the preclinical evaluation via oral administration. Preformulation studies including physical form screening and physicochemical properties determination were performed. Polymorphism was observed, and the more thermodynamically stable form was selected for further studies. ZL006 showed certain supersaturation solubility, although the thermodynamic solubility in FaSSIF was low, which indicated the supersaturating formulation might work. Parameter sensitivity analysis by in silico simulation predicted that in vivo exposure was sensitive to solubility, while particle size reduction would have limited impact on exposure. Based on in silico prediction and the understanding of the molecule from preformulation studies, solid dispersion approach was selected. A preliminary dose escalation pharmacokinetic study in rats demonstrated that in vivo exposure increased in dose-proportional manner from 12.5 mg/kg to 50 mg/kg with around 50% oral bioavailability after oral dosing of the solid dispersion. This work showed that combination of preformulation studies and in silico simulation could efficiently guide the selection of enabling formulation, which could save resources at preclinical stage.

Investigating the Mechanism of L-Valine in Improving the Stability of Gabapentin Combining Chemical Analysis Experiments with Computational Pharmacy.

The mechanism of L-Val on how to improve the stability of gabapentin (GBP) was described by the combination of chemical analysis experiments and computer simulations. Scanning electron microscope (SEM), powder X-ray diffraction (PXRD), and differential scanning calorimeter (DSC), coupled with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), were used to identify ß-GBP prepared by rapid solvent removal method. The reaction barriers on crystal planes, ß-GBP (100) and ß-GBP (10-1), are smaller than α-GBP and γ-GBP, reaching 276.65 kJ/mol and 299.57 kJ/mol, respectively. Thus, it was easier for ß-GBP to form lactam, and the occurrence of ß-GBP would lead the worse stability of α-GBP. The addition of neutral amino acids such as L-Val could improve the stability of α-GBP effectively. The adsorption energy of α-GBP (002) crystal plane with L-Val is larger than that of other crystal planes, reaching 42.17 kJ/mol. Hydrogen bond was the combination of L-Val and GBP main crystal planes, which could inhibit the crystal transformation of α-GBP. These results suggest that neutral amino acid protectants, such as L-Val, could improve the stability of α-GBP effectively, and inhibition of crystal transformation is one of the effective methods to improve the stability of polymorphic drugs.

Optimization of Extended-Release ZL-004 Nanosuspensions for In Vivo Pharmacokinetic Study to Enhance Low Solubility and Compliance.

ZL-004, a promising small molecule that increases white blood cell counts, was developed for extended-release nanosuspensions to improve low solubility and compliance of patients. In vivo pharmacokinetic studies of nanosuspensions with different particle sizes and administration volumes were conducted. Unexpectedly, Cmax of NS-PC-L (1156 nm) was 1.3 fold higher than NS-PB-L (836 nm), and area under plasma concentration-time curve (AUC) was similar. It suggested that in vivo behavior of nanosuspensions was influenced significantly by the original dissolved drug, which did not only rely on the particle size but also the amount of the free stabilizers. In addition, smaller administration volume (0.1 mL) achieved significantly lower Cmax and AUC than the higher volume (0.5 mL), due to the reduced amount of dissolved drug. DSC and XPRD demonstrated that the crystal forms of nanosuspensions prepared by the precipitation method and high-pressure homogenization were similar; therefore, in vivo behaviors did not show significant differences. An additional 0.15% PEG 4000 enhanced the redispersity and maintained the particle size for 3 months. Finally, a nanosuspensions with the desired initial release was achieved, which lasted approximately 32 days steadily after a single dose. AUC and t1/2 were 161.2 fold and 22.9 fold higher than oral administration.

Zinc pthalocyanine-loaded chitosan/mPEG-PLA nanoparticles-mediated photodynamic therapy for the treatment of cutaneous squamous cell carcinoma.

Zinc pthalocyanine (ZnPc) is a second-generation photodynamic therapy (PDT) sensitizer with sufficient PDT activity for squamous cell carcinoma (SCC). ZnPc is hydrophobic and insoluble in water, which creates hurdles in systemic administration and hence restricts its use in clinic. Here we have loaded ZnPc on chitosan/methoxy polyethylene glycol-polylactic acid (CPP) nanoparticles to form Z-CPP to enhance PDT efficacy. In vitro and in vivo studies were performed to see dark toxicity of the compounds ZnPc, CPP and Z-CPP. Then PDT was done and its growth inhibitory effect on SCC cells was evaluated. In addition, reactive oxygen species (ROS) formation and apoptosis of cancer cells following PDT were studied. The results showed that the tested compounds exhibit no dark toxicity and the effect of PDT was significantly better with Z-CPP when compared to free ZnPc (P < .05). Photoactivation of Z-CPP led to a dose-dependent growth inhibition of cancer cells of >50% at 1 µM to >80% at 10 µM concentration. Also Z-CPP-treated cells had highest number of apoptotic cells and produced more ROS compared to free ZnPc-treated cells (P < .05). Hence, this study suggests that Z-CPP is a suitable pharmaceutical compound to increase PDT efficacy.

Transcutaneous immunization via rapidly dissolvable microneedles protects against hand-foot-and-mouth disease caused by enterovirus 71.

Recent large outbreaks of hand-foot-and-mouth disease (HFMD) have seriously affected the health of young children. Enterovirus 71 (EV71) is the main causative agent of HFMD. Herein, for the first time, rapidly dissolvable microneedles (MNs) loaded with EV71 virus-like particles (VLPs) were evaluated whether they could induce robust immune responses that confer protection against EV71 infection. The characteristics of prepared MNs including hygroscopy, mechanical strength, insertion capacity, dissolution profile, skin irritation and storage stability were comprehensively assessed. EV71 VLPs remained morphologically stable during fabrication. The MNs made of sodium hyaluronate maintained their insertion ability for at least 3h even at a high relative humidity of 75%. With the aid of spring-operated applicator, EV71 MNs (approximately 500µm length) could be readily penetrated into the mouse skin in vivo, and then rapidly dissolved to release encapsulated antigen within 2min. Additionally, MNs induced slight erythema that disappeared within a few hours. More importantly, mouse immunization and virus challenge studies demonstrated that MNs immunization induced high level of antibody responses conferring full protection against lethal EV71 virus challenge that were comparable to conventional intramuscular injection, but with only 1/10th of the delivered antigen (dose sparing). Consequently, our rapidly dissolving MNs may present as an effective and promising transcutaneous immunization device for HFMD prophylaxis among children.

Ursolic Acid Nanocrystals for Dissolution Rate and Bioavailability Enhancement: Influence of Different Particle Size.

BACKGROUND: Ursolic acid (UA), a natural pentacyclic triterpenoid, has been reported to possess a variety of pharmacological activities, but the poor oral bioavailability of UA owing to the poor aqueous solubility and membrane permeability limits the further clinical application. OBJECTIVE: The purpose of the present study was to develop UA nanocrystals and microcrystals employing high pressure homogenization (HPH) and to evaluate their effects on UA oral bioavailability. METHOD: The crystalline morphology of UA nanocrystals and microcrystals prepared by HPH was observed by scanning electron microscopy and the crystalline state was characterized by differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). The dissolution rate of UA nanocrystals in different pH conditions was tested. Oral bioavailability of UA nanocrystals and microcrystals comparing with UA coarse suspension was evaluated in SD rats after 50 mg·kg-1 administration. RESULTS: UA nanocrystals and microcrystals, the size of which ranged between 291.7 nm and 1299.3 nm were obtained. The results of DSC and PXRD revealed a degree of crystalline-amorphous transformation during HPH preparation. A significant increase was observed in the dissolution rate of UA nanocrystals. The relative bioavailability of UA nanocrystals and microcrystals exhibited 2.56 and 1.40-fold enhancement than that of UA coarse suspension, respectively, along with an increased peak concentration and a prolonged retention. CONCLUSION: The nanosized UA crystal is a viable and efficient approach to improve the oral bioavailability of UA.

Treating cutaneous squamous cell carcinoma using 5-aminolevulinic acid polylactic-co-glycolic acid nanoparticle-mediated photodynamic therapy in a mouse model.

BACKGROUND: Squamous cell carcinoma (SCC) is a common skin cancer, and its treatment is still difficult. The aim of this study was to evaluate the effectiveness of nanoparticle (NP)-assisted 5-aminolevulinic acid (ALA) delivery for topical photodynamic therapy (PDT) of cutaneous SCC. MATERIALS AND METHODS: Ultraviolet-induced cutaneous SCCs were established in hairless mice. ALA-loaded polylactic-co-glycolic acid (PLGA) NPs were prepared and characterized. The kinetics of ALA PLGA NP-induced protoporphyrin IX fluorescence in SCCs, therapeutic efficacy of ALA NP-mediated PDT, and immune responses were examined. RESULTS: PLGA NPs enhanced protoporphyrin IX production in SCC. ALA PLGA NP-mediated topical PDT was more effective than free ALA of the same concentration in treating cutaneous SCC. CONCLUSION: PLGA NPs provide a promising strategy for delivering ALA in topical PDT of cutaneous SCC.

Rapidly dissolvable microneedle patches for transdermal delivery of exenatide.

PURPOSE: To assess the feasibility of transdermal delivery of exenatide (EXT) using low-molecular-weight sodium hyaluronate (HA) dissolving microneedles (MNs) patches for type 2 diabetes mellitus therapy. METHODS: Micromold casting method was used to fabricate EXT-loaded dissolving MNs. The characteristics of prepared MNs including mechanical strength, in vitro/in vivo insertion capacity, dissolution profile and storage stability were then investigated. Finally, the in vivo pharmacokinetics and hypoglycemic effects were compared with traditional subcutaneous (SC) injection. RESULTS: EXT-loaded dissolving MNs made of HA possessed sufficient mechanical strength and the strength could be weakened as the water content increases. The EXT preserved its pharmacological activity during fabrication and one-month storage. With the aid of spring-operated applicator, dissolving MNs could be readily penetrated into the skin in vitro/in vivo, and then rapidly dissolved to release encapsulated drug within 2 min. Additionally, transepidermal water loss (TEWL) determinations showed that skin's barrier properties disrupted by MNs recovered within 10-12 h. Transdermal pharmacokinetics and antidiabetic effects studies demonstrated that fabricated EXT MNs induced comparable efficacy to SC injection. CONCLUSIONS: Our rapidly dissolving MNs patch appears to an excellent, painless alternative to conventional SC injection of EXT, and this minimally invasive device might also be suitable for other biotherapeutics.

In vitro evaluation of 5-aminolevulinic acid (ALA) loaded PLGA nanoparticles.

BACKGROUND: 5-Aminolevulinic acid (ALA) is a prodrug for topical photodynamic therapy. The effectiveness of topical ALA can be limited by its bioavailability. The aim of this study was to develop a novel ALA delivery approach using poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). METHODS: A modified double emulsion solvent evaporation method was used to prepare ALA loaded PLGA NPs (ALA PLGA NPs). The characteristics, uptake, protoporphyrin IX fluorescence kinetics, and cytotoxicity of ALA PLGA NPs toward a human skin squamous cell carcinoma cell line were examined. RESULTS: The mean particle size of spherical ALA PLGA NPs was 65.6 nm±26 nm with a polydispersity index of 0.62. The encapsulation efficiency was 65.8%±7.2% and ALA loading capacity was 0.62%±0.27%. When ALA was dispersed in PLGA NPs, it turned into an amorphous phase. ALA PLGA NPs could be taken up by squamous cell carcinoma cells and localized in the cytoplasm. The protoporphyrin IX fluorescence kinetics and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay showed that ALA PLGA NPs were more effective than free ALA of the same concentration. CONCLUSION: PLGA NPs provide a promising ALA delivery strategy for topical ALA-photodynamic therapy of skin squamous cell carcinoma.