Computationally guided high-throughput design of self-assembling drug nanoparticles.

Nanoformulations of therapeutic drugs are transforming our ability to effectively deliver and treat a myriad of conditions. Often, however, they are complex to produce and exhibit low drug loading, except for nanoparticles formed via co-assembly of drugs and small molecular dyes, which display drug-loading capacities of up to 95%. There is currently no understanding of which of the millions of small-molecule combinations can result in the formation of these nanoparticles. Here we report the integration of machine learning with high-throughput experimentation to enable the rapid and large-scale identification of such nanoformulations. We identified 100 self-assembling drug nanoparticles from 2.1 million pairings, each including one of 788 candidate drugs and one of 2,686 approved excipients. We further characterized two nanoparticles, sorafenib-glycyrrhizin and terbinafine-taurocholic acid both ex vivo and in vivo. We anticipate that our platform can accelerate the development of safer and more efficacious nanoformulations with high drug-loading capacities for a wide range of therapeutics.

In Vitro Sensitivity Analysis of the Gastrointestinal Dissolution Profile of Weakly Basic Drugs in the Stomach-to-Intestine Fluid Changing System: Explanation for Variable Plasma Exposure after Oral Administration.

An in vitro methodology for simulating the change in the pH and composition of gastrointestinal fluid associated with the transition of orally administered drugs from the stomach to the small intestine was developed (the stomach-to-intestine fluid changing system (the SIFC system)). This system was applied to in vitro sensitivity analysis on the dissolution of weakly basic drugs, and the obtained results were discussed in relation to the intrasubject variability in the plasma exposure in human bioequivalence (BE) study. Three types of protocols were employed (steep pH change: pH 1.6 FaSSGF → pH 6.5 FaSSIF, gradual pH change: pH 1.6 FaSSGF → pH 6.5 FaSSIF, and high gastric pH: pH 4.0 FaSSGF → pH 6.5 FaSSIF). Regardless of the protocols and the forms of drug applied in active pharmaceutical ingredient powder or formulation, dissolution profiles of pioglitazone after fluid shift were similar and the final concentrations in FaSSIF were approximately equal to the saturation solubility in FaSSIF, supporting its small intrasubject variance in human BE study. In contrast, dissolved concentration of terbinafine in the SIFC system became less than half in the high gastric pH protocol than that in other protocols, suggesting the fluctuation of gastric pH as one of the factors of high intrasubject variance of terbinafine in human. Plasma exposure of telmisartan was highly variable especially at the high dose. Although the dissolution of telmisartan in the SIFC system was greatly improved by formulation, it considerably fluctuated during fluid shift especially at the high dose, which corresponds well to in vivo results.

Lysinibacillus Isolate MK212927: A Natural Producer of Allylamine Antifungal 'Terbinafine'.

Resistance to antifungal agents represents a major clinical challenge, leading to high morbidity and mortality rates, especially in immunocompromised patients. In this study, we screened soil bacterial isolates for the capability of producing metabolites with antifungal activities via the cross-streak and agar cup-plate methods. One isolate, coded S6, showed observable antifungal activity against Candida (C.) albicans ATCC 10231 and Aspergillus (A.) niger clinical isolate. This strain was identified using a combined approach of phenotypic and molecular techniques as Lysinibacillus sp. MK212927. The purified metabolite displayed fungicidal activity, reserved its activity in a relatively wide range of temperatures (up to 60 °C) and pH values (6-7.8) and was stable in the presence of various enzymes and detergents. As compared to fluconazole, miconazole and Lamisil, the minimum inhibitory concentration of the metabolite that showed 90% inhibition of the growth (MIC90) was equivalent to that of Lamisil, half of miconazole and one fourth of fluconazole. Using different spectroscopic techniques such as FTIR, UV spectroscopy, 1D NMR and 2D NMR techniques, the purified metabolite was identified as terbinafine, an allylamine antifungal agent. It is deemed necessary to note that this is the first report of terbinafine production by Lysinibacillus sp. MK212927, a fast-growing microbial source, with relatively high yield and that is subject to potential optimization for industrial production capabilities.

Targeting pathogenic fungi, bacteria and fungal-bacterial biofilms by newly synthesized quaternary ammonium derivative of pyridoxine and terbinafine with dual action profile.

Many pathogenic bacteria and microscopic fungi form rigid polymicrobial biofilms this way enhancing their resistant to treatment. A series of novel pyridoxine-based quaternary ammonium derivatives of terbinafine characterized by both antifungal and antibacterial activities was designed. The leading compound named KFU-127 exhibits promising antifungal and antibacterial activities against various bacteria and micromycetes in both planktonic and biofilm-embedded forms demonstrating MIC values comparable with those of conventional antifungals and antimicrobials. Similar to other antiseptics like benzalkonium chloride and miramistin, KFU-127 is considerably toxic for eukaryotic cells that limits is application to topical treatment options. On the other hand, KFU-127 reduces the number of viable biofilm-embedded bacteria and C. albicans by 3 orders of magnitude at concentrations 2-4 times lower than those of reference drugs and successfully eradicates S. aureus-C. albicans mixed biofilms. The mechanism of antimicrobial action of KFU-127 is bimodal including both membrane integrity damage and pyridoxal-dependent enzymes targeting. We expect that this bilateral mechanism would result in lower rates of resistance development in both fungal and bacterial pathogens. Taken together, our data suggest KFU-127 as a new promising broad spectrum topical antimicrobial capable of one-shot targeting of bacterial and fungal-bacterial biofilms.

FT-IR investigation of Terbinafine interaction with stratum corneum constituents.

Terbinafine (Tbf) is a well-established anti-fungal agent used for management of a variety of dermal conditions including ringworm and athlete's foot. Both the biochemical mechanism of Tbf fungicidal action (based on squalene epoxidase inhibition) and the target region for Tbf in vivo (the stratum corneum (SC)) are well determined. However, the biochemical and pharmacokinetic approaches used to evaluate Tbf biochemistry provide no biophysical information about molecular level physical changes in the SC upon Tbf binding. Such information is necessary for improved drug and formulation design. IR spectroscopic methods were used to evaluate the effects of Tbf on keratin structure in environments commonly used in pharmaceutics to mimic those in vivo. The Amide I and II spectral regions (1500-1700 cm-1) provided an effective means to monitor keratin secondary structure changes, while a Tbf spectral feature near 775 cm-1 provides a measure of relative Tbf levels in skin. Interaction of Tbf with the SC induced substantial ß-sheet formation in the keratin, an effect which was partially reversed both by ethanol washing and by exposure to high relative humidity. The irreversibility suggests the presence of a Tbf reservoir (consistent with kinetic studies), permitting the drug to be released in a controlled manner into the surrounding tissue.

Increasing Susceptibility of Drug-Resistant Candida albicans to Fluconazole and Terbinafine by 2(5H)-Furanone Derivative.

The frequency of mycoses caused by drug-resistant fungal pathogen Candida albicans has increased drastically over the last two decades. The spread of drug-resistant strains, along with the limitations of currently available antifungals, complicates the management of fungal infections, thereby representing great challenges for clinical healthcare. Among various antimicrobial pharmacophores, 2(5H)-furanone derivatives have demonstrated antimicrobial, antifungal, and antibiofilm activities. In this study, we report the antifungal activity of the 2(5H)-furanone derivative F105, consisting of three pharmacophores, namely chlorinated 2(5H)-furanone, sulfonyl group, and l-menthol moiety. Although exhibiting moderate antifungal activity alone with the minimum inhibitory concentration (MIC) values of 32-256 µg/mL, F105 potentiates the activity of fluconazole and terbinafine with fractional inhibitory concentration index (FICI) values of 0.27-0.50. Thus, 16 µg/mL of F105 reduced the MICs of these antifungals against fluconazole-resistant C. albicans isolates four-fold, achieving similar values as for the intermediately susceptible phenotype. Confocal laser scanning microscopy revealed that the fluorescent 2(5H)-furanone derivative F145 was also able to penetrate through biofilms formed by C. albicans. Indeed, in the presence of F105, even sub-MIC concentrations of both fluconazole and terbinafine led to significant reduction of C. albicans CFUs in the mature biofilm. Thus, F105 appears to be a promising candidate for the development of novel antifungal agents as well as enhancers of current antifungal agents, particularly for the treatment of drug-resistant C. albicans infections.

Terbinafine-loaded branched PLGA-based cationic nanoparticles with modifiable properties.

Although the systemic administration of terbinafine is quite well tolerated, topical treatment of the local infections is often preferred. New formulation strategies in topical antifungal therapy represent the polymeric nanoparticles (NPs). We successfully employed the originally synthesized PLGA derivatives of branched architectures of various molar masses, branching ratio, and high number of terminal hydroxyl or carboxyl groups for compounding of terbinafine loaded nanoparticles by nanoprecipitation method. Employing the polymers with tailored properties allowed us to formulate the NPs with desired particle size, loading capacity for drug, mucoadhesive properties, and drug release profile. The hydrophobicity and the polyester concentration revealed the main impact on the NPs size ranging from 100 to 600 nm. The stability of the nanosuspension is demonstrated by zeta potential >25 mV, and polydispersity index values <0.2. We used terbinafine in its less dissolved form of the base to increase the drug loading and delay the release. Cationic surfactant as stabilizer give the NPs high positive surface charge enhancing the adhesion to the mucosal surfaces. All formulations provided prolonged sustained release of terbinafine for several days. Antimicrobial potential has been proven by agar-well diffusion method.

Determination of terbinafine in human plasma using UPLC-MS/MS: Application to a bioequivalence study in healthy subjects.

A high-throughput and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of terbinafine in human plasma. The method employed liquid-liquid extraction of terbinafine and terbinafine-d7 (used as internal standard) from 100 µL human plasma with ethyl acetate-n-hexane (80:20, v/v) solvent mixture. Chromatography was performed on a BEH C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile-8.0 mm ammonium formate, pH 3.5 (85:15, v/v) under isocratic elution. For quantitative analysis, MS/MS ion transitions were monitored at m/z 292.2/141.1 and m/z 299.1/148.2 for terbinafine and terbinafine-d7, respectively, using electrospray ionization in the positive mode. The method was validated according to regulatory guidance for selectivity, sensitivity, linearity, recovery, matrix effect, stability, dilution reliability and ruggedness with acceptable accuracy and precision. The method shows good linearity over the tested concentration range from 1.00 to 2000 ng/mL (r2 ≥ 0.9984). The intra-batch and inter-batch precision (CV) was 1.8-3.2 and 2.1-4.5%, respectively. The method was successfully applied to a bioequivalence study with 250 mg terbinafine in 32 healthy subjects. The major advantage of this method includes higher sensitivity, small plasma volume for processing and a short analysis time.

CYP2C19 and 3A4 Dominate Metabolic Clearance and Bioactivation of Terbinafine Based on Computational and Experimental Approaches.

Lamisil (terbinafine) is an effective, widely prescribed antifungal drug that causes rare idiosyncratic hepatotoxicity. The proposed toxic mechanism involves a reactive metabolite, 6,6-dimethyl-2-hepten-4-ynal (TBF-A), formed through three N-dealkylation pathways. We were the first to characterize them using in vitro studies with human liver microsomes and modeling approaches, yet knowledge of the individual enzymes catalyzing reactions remained unknown. Herein, we employed experimental and computational tools to assess terbinafine metabolism by specific cytochrome P450 isozymes. In vitro inhibitor phenotyping studies revealed six isozymes were involved in one or more N-dealkylation pathways. CYP2C19 and 3A4 contributed to all pathways, and so, we targeted them for steady-state analyses with recombinant isozymes. N-Dealkylation yielding TBF-A directly was catalyzed by CYP2C19 and 3A4 similarly. Nevertheless, CYP2C19 was more efficient than CYP3A4 at N-demethylation and other steps leading to TBF-A. Unlike microsomal reactions, N-denaphthylation was surprisingly efficient for CYP2C19 and 3A4, which was validated by controls. CYP2C19 was the most efficient among all reactions. Nonetheless, CYP3A4 was more selective at steps leading to TBF-A, making it more effective in terbinafine bioactivation based on metabolic split ratios for competing pathways. Model predictions did not extrapolate to quantitative kinetic constants, yet some results for CYP3A4 and CYP2C19 agreed qualitatively with preferred reaction steps and pathways. Clinical data on drug interactions support the CYP3A4 role in terbinafine metabolism, while CYP2C19 remains understudied. Taken together, knowledge of P450s responsible for terbinafine metabolism and TBF-A formation provides a foundation for investigating and mitigating the impact of P450 variations in toxic risks posed to patients.

Photostability of Terbinafine Under UVA Irradiation: The Effect of UV Absorbers.

The photostability of drugs administered topically on unprotected skin is a complex phenomenon that could be connected with the loss of activity or, rather rarely, the occurrence of toxic degradation products. In this study, an in-depth investigation of the photostability of terbinafine, in both solutions and formulations, was conducted, taking into account the presence of UV absorbers such as TiO2 , ZnO, avobenzone, 3-(4-methylbenzylidene)camphor, octocrylene, benzophenone-1 and benzophenone-2. The clear photocatalytic degradation of terbinafine in ethanol solution was observed in the presence of TiO2 and/or ZnO. In other cases, terbinafine was stable, with the exception of, in the presence of octocrylene. The presumed degradation products of terbinafine were identified for the first time using LC/MS/MS, and transformation pathways were proposed. In the case of a cream formulation, the percentage of initial terbinafine content was almost unchanged in the presence of the UV absorbers benzophenone-1, benzophenone-2 and 3-(4-methylbenzylidene)camphor. In vitro cytotoxicity risk assessment of terbinafine based on photostability under UVA irradiation was evaluated using the human skin fibroblast BJ (ATCC® CRL-2522™), and this showed no statistically significant difference in cell viability for all samples analyzed.

Criteria for the selection of switch OTC drugs based on patient benefits, efficacy, and safety [II]: Comparing the physicochemical and pharmaceutical properties of brand-name and switch OTC terbinafine hydrochloride cream.

The physicochemical properties (pH, yield value, and squeeze force) of a drug for dermatomycosis, a terbinafine hydrochloride-containing cream (a brand-name product), and 12 over-the-counter drugs (OTCs) were measured and compared to ascertain the characteristics of each product. The pH of the brand-name product, Lamisil, was 4.1, and that of the OTC products ranged from 4.2 to 7.6; Lamisil Plus (7.6) had a significantly higher pH. Moreover, the yield value for Lamisil, as an index of cream ductility, was 128 dyn/cm2, and that for the OTC products ranged from 110 to 887 dyn/cm2. In particular, the OTC products Damalin (887 dyn/cm2), Barriact (512 dyn/cm2), and Exiv Deep (663 dyn/cm2) had a significantly higher yield value. In addition, the squeeze force was measured by attaching a HapLog® to the thumb and second finger. The squeeze force for Lamisil was 12.9 N, and that for the OTC products ranged from 1.8 to 14.6 N. The OTC product Bilumon (1.8 N) had a significantly lower squeeze force. These results indicated that there were marked differences in the pharmaceutical properties of brand-name and OTC products. External preparations are characterized by their feel during use. Based on the current results, the pharmaceutical characteristics of drugs resulted in differences in their feel during use, suggesting that products appropriate for individual patients can be recommended.

Lamisil (terbinafine) toxicity: Determining pathways to bioactivation through computational and experimental approaches.

Lamisil (terbinafine) may cause idiosyncratic liver toxicity through a proposed toxicological mechanism involving the reactive metabolite 6,6-dimethyl-2-hepten-4-ynal (TBF-A). TBF-A toxicological relevance remains unclear due to a lack of identification of pathways leading to and competing with TBF-A formation. We resolved this knowledge gap by combining computational modeling and experimental kinetics of in vitro hepatic N-dealkylation of terbinafine. A deep learning model of N-dealkylation predicted a high probability for N-demethylation to yield desmethyl-terbinafine followed by N-dealkylation to TBF-A and marginal contributions from other possible pathways. We carried out steady-state kinetic experiments with pooled human liver microsomes that relied on development of labeling methods to expand metabolite characterization. Those efforts revealed high levels of TBF-A formation and first order decay during metabolic reactions; actual TBF-A levels would then reflect the balance between those processes as well as reflect the impact of stabilizing adduction with glutathione and other biological molecules. Modeling predictions and experimental studies agreed on the significance of N-demethylation and insignificance of N-denaphthylation in terbinafine metabolism, yet differed on importance of direct TBF-A formation. Under steady-state conditions, the direct pathway was the most important source of the reactive metabolite with a Vmax/Km of 4.0 pmol/min/mg protein/µM in contrast to model predictions. Nevertheless, previous studies show that therapeutic dosing leads to accumulation of desmethyl-terbinafine in plasma, which means that likely sources for TBF-A would draw from metabolism of both the major metabolite and parent drug based on our modeling and experimental studies. Through this combination of novel modeling and experimental approaches, we are the first to identify pathways leading to generation of TBF-A for assessing its role in idiosyncratic adverse drug interactions.

The role of HLA-A*33:01 in patients with cholestatic hepatitis attributed to terbinafine.

BACKGROUND & AIMS: Terbinafine is an antifungal agent that has been associated with rare instances of hepatotoxicity. In this study we aimed to describe the presenting features and outcomes of patients with terbinafine hepatotoxicity and to investigate the role of human leukocyte antigen (HLA)-A*33:01. METHODS: Consecutive high causality cases of terbinafine hepatotoxicity enrolled into the Drug Induced Liver Injury Network were reviewed. DNA samples underwent high-resolution confirmatory HLA sequencing using the Ilumina MiSeq platform. RESULTS: All 15 patients with terbinafine hepatotoxicity were more than 40 years old (median = 57 years), 53% were female and the median latency to onset was 38 days (range 24 to 114 days). At the onset of drug-induced liver injury, 80% were jaundiced, median serum alanine aminotransferase was 448 U/L and alkaline phosphatase was 333 U/L. One individual required liver transplantation for acute liver failure during follow-up, and 7 of the 13 (54%) remaining individuals had ongoing liver injury at 6 months, with 4 demonstrating persistently abnormal liver biochemistries at month 24. High-resolution HLA genotyping confirmed that 10 of the 11 (91%) European ancestry participants were carriers of the HLA-A*33:01, B*14:02, C*08:02 haplotype, which has a carrier frequency of 1.6% in European Ancestry population controls. One African American patient was also an HLA-A*33:01 carrier while 2 East Asian patients were carriers of a similar HLA type: A*33:03. Molecular docking studies indicated that terbinafine may interact with HLA-A*33:01 and A*33:03. CONCLUSIONS: Patients with terbinafine hepatotoxicity most commonly present with a mixed or cholestatic liver injury profile and frequently have residual evidence of chronic cholestatic injury. A strong genetic association of HLA-A*33:01 with terbinafine drug-induced liver injury was confirmed amongst Caucasians. LAY SUMMARY: A locus in the human leukocyte antigen gene (HLA-A*33:01, B*14:02, C*08:02) was significantly overrepresented in Caucasian and African American patients with liver injury attributed to the antifungal medication, terbinafine. These data along with the molecular docking studies demonstrate that this genetic polymorphism is a plausible risk factor for developing terbinafine hepatotoxicity and could be used in the future to help doctors make a diagnosis more rapidly and confidently.

Two strategies to enhance ungual drug permeation from UV-cured films: Incomplete polymerisation to increase drug release and incorporation of chemical enhancers.

UV-curable gels, which polymerise into long-lasting films upon exposure to UVA, have been identified as potential topical drug carriers for the treatment of nail diseases. Limitations of such films include incomplete drug release and low ungual drug permeation. The aim of the work herein was therefore to investigate two strategies, namely: (1) increasing drug release from the film, and (2) increasing nailplate permeability, with the ultimate goal of enhancing ungual drug permeation. To increase drug release via Strategy 1, a UV-LED lamp (whose emitted light was suboptimal for gel polymerisation) was used, and it was hypothesised that such a lamp would result in films that are less polymerised/cross-linked and where the drugs are less 'trapped'. Indeed, the suboptimal lamp influenced polymerisation, such that the films were thinner, had lower glass transition temperatures and enabled a slightly greater (by 15%) drug release of one of the two drugs tested. However, the greater drug release had only a modest impact on ungual drug permeation. To evaluate Strategy 2, i.e. increase nailplate permeability, chemical ungual enhancers, 2-mercaptoethanol (ME), 2-methyl pyrrolidone (NMP), PEG 200 and water were incorporated within the UV-cured films. These chemicals caused increased ungual drug permeation, with ME showing the greatest (by 140%), and water showing the least (by 20%) increase in the amount of drug permeated by day 30. Surprisingly, these chemicals also caused increased drug release from the films, with ME once again having the greatest effect (by 51%) and water the least effect (by 12%). It seems that these chemicals were increasing ungual drug permeation via their influence on drug release (i.e. via their impact on the film) as well as via their influence on the nail itself. We conclude that, of the two strategies tested, the second strategy proved to be more successful at enhancing ungual drug permeation.

Generic selection criteria for safety and patient benefit [VII]: Comparing the physicochemical and pharmaceutical properties of brand-name and generic terbinafine hydrochloride cream.

We measured and compared the physicochemical properties (pH, yield value, and squeeze force) of a drug for dermatomycosis, terbinafine hydrochloride-containing cream (brand-name product), and 12 generic products to clarify the characteristics of each product. On pH measurement, the pH value of the brand-name product, Lamisil, was 4.8, and those of the generic products ranged from 4.3 to 5.5, showing no marked difference. Furthermore, the yield value of Lamisil, as an index of cream ductility, was 122.2 dyn/cm2, and those of the generic products ranged from 42.1 to 1,621.5 dyn/cm2. In particular, the value of a generic product, Taiyo (42.1 dyn/cm2), was significantly lower, whereas that of another one, Viras (1,621.0 dyn/cm2), was significantly higher. In addition, the squeeze force was measured by attaching a HapLog® to the thumb and second finger. The value of Lamisil was 12.9 N, and those of the generic products ranged from 8.0 to 15.4 N. The values of generic products, Mylan (8.6 N), Tebinaceil (9.0 N), and Kelger (8.0 N), were significantly lower, whereas that of another one, Viras (15.4 N), was significantly higher. These results showed that there were marked differences in the pharmaceutical properties between the generic and brand-name products. The above pharmaceutical characteristics of drugs facilitated the presentation of reasons for differences in the sense of use, which characterizes external preparations, suggesting that products appropriate for individual patients can be recommended.

High-level recombinant production of squalene using selected Saccharomyces cerevisiae strains.

For recombinant production of squalene, which is a triterpenoid compound with increasing industrial applications, in microorganisms generally recognized as safe, we screened Saccharomyces cerevisiae strains to determine their suitability. A strong strain dependence was observed in squalene productivity among Saccharomyces cerevisiae strains upon overexpression of genes important for isoprenoid biosynthesis. In particular, a high level of squalene production (400 ± 45 mg/L) was obtained in shake flasks with the Y2805 strain overexpressing genes encoding a bacterial farnesyl diphosphate synthase (ispA) and a truncated form of hydroxyl-3-methylglutaryl-CoA reductase (tHMG1). Partial inhibition of squalene epoxidase by terbinafine further increased squalene production by up to 1.9-fold (756 ± 36 mg/L). Furthermore, squalene production of 2011 ± 75 or 1026 ± 37 mg/L was obtained from 5-L fed-batch fermentations in the presence or absence of terbinafine supplementation, respectively. These results suggest that the Y2805 strain has potential as a new alternative source of squalene production.