Effect of interactive ternary mixtures on dispersion characteristics of ipratropium bromide in dry powder inhaler formulations

The purpose of this investigation was to evaluate the effect of mixing order and the influence of adding fines on in vitro performance of ipratropium bromide (ITB) dry powder inhaler formulations. Coarse lactose (CL) in varying mass ratio with or without addition of micronized lactose (ML) and ITB in different mixing sequences was used to formulate ternary mixtures. A binary mixture composed of CL and ITP served as control. The in vitro deposition of ITB from these formulations was measured using an Andersen cascade impactor (aerosolization at 39 L/min) employing a HandiHaler as the delivery device. It was observed that mixing order has a significant effect (P<.05) on in vitro deposition of ITB. Formulations with preblending of CL and ITB produced similar deposition profiles as the control, regardless of the added ML. In contrast, formulations without preblending resulted in significantly higher fine particle dose (FPD) as compared with the control. In addition, an increased quantity of ML generally resulted in an increase in drug deposition. The results show that the effect of ML on dispersion of ITB is highly dependent upon the mixing order. The evaluation of atomic force measurement (AFM) to forecast drug detachment and predict the aerodynamic characteristics resulted in similar attraction forces for the different pairs lactose/lactose (42.66±25.01 nN) and lactose/ITB (46.77±17.04 nN). Published: April 20, 2007     

Industrializing AI/ML during the end-to-end drug discovery process

Drug discovery aims to select proper targets and drug candidates to address unmet clinical needs. The end-to-end drug discovery process includes all stages of drug discovery from target identification to drug candidate selection. Recently, several artificial intelligence and machine learning (AI/ML)-based drug discovery companies have attempted to build data-driven platforms spanning the end-to-end drug discovery process. The ability to identify elusive targets essentially leads to the diversification of discovery pipelines, thereby increasing the ability to address unmet needs. Modern ML technologies are complementing traditional computer-aided drug discovery by accelerating candidate optimization in innovative ways. This review summarizes recent developments in AI/ML methods from target identification to molecule optimization, and concludes with an overview of current industrial trends in end-to-end AI/ML platforms.     

Cytosolic delivery of macromolecules. II. Mechanistic studies with pH-sensitive morpholine lipids

Drug carriers containing weak acids or bases can promote cytosolic delivery of macromolecules by exploiting the acidic pH of the endosome. We have prepared two pH-sensitive mono-stearoyl derivatives of morpholine, one with a (2-hydroxy) propylene (ML1) linker and the other, an ethylene (ML2) linker. The pK(a) values of lipids ML1 and ML2, when incorporated into liposomes, are 6.12 and 5.91, respectively. Both lipids disrupt human erythrocytes at pH equal to or below their pK(a) but show no such activity at pH 7.4. Confocal microscopy studies suggest partial endosome-to-cytosol transfer of fluorescent dextran (MW 10 kDa) encapsulated in liposomes that contained 20 mol% of morpholine lipids. Interestingly, co-incubation of morpholine lipids in free or micellar form (without liposomal incorporation) with dextran resulted in efficient cytosolic delivery. Upon acidification to the endosomal pH, liposomes containing ML1 revealed: (a). leakage of entrapped solute that is independent of solute size; (b). lack of liposomal collapse into micelles as evidenced by photon correlation spectroscopy and UV light scattering; and (c). minimal inter-bilayer interactions as shown in a fluorescence resonance energy transfer assay. These observations are consistent with progressive intravesicular reorganization of lipids into stable liposomes of smaller size, but of more homogeneous distribution, upon acidification. The results emphasize a need to manipulate liposomal formulations containing ML1 such that ML1 will promote catastrophic collapse of liposomes to mixed micelles upon exposure to acidic pH. It is only then that micelle-mediated permeabilization of the endosomal membrane will lead to efficient cytosolic delivery of macromolecules originally loaded in liposomes.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci.

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Trichinella spiralis: major histocompatibility complex-associated elimination of encysted muscle larvae in swine

A heretofore undescribed host-mediated reactivity against encapsulated muscle larvae (ML) of the nematode Trichinella spiralis is reported. Inbred miniature swine (NIH minipigs) of three independent SLA phenotypes, which received a primary oral dose of 300 T. spiralis ML, successfully resisted a secondary infection of 10,000 ML; however, only pigs of the SLAa/a phenotype exhibited an unusual and highly significant reduction in the numbers of encysted ML from the primary infection (P less than 0.0003). This initial anti-encysted ML reactivity was confirmed in subsequent trials by comparing the prechallenge ML burdens with the reduced ML numbers in primary-infected aa pigs after challenge. Analyses of inbred strains of mice, selected for major histocompatibility type and for resistance or susceptibility to infection with T. spiralis, showed no such anti-encysted ML response. Because elimination of encysted T. spiralis ML had been accomplished previously only through selected drug regimens, our demonstration of a nonpharmacological, host-mediated reactivity against this stage of the parasite in swine highlights the importance of MHC genes in regulating disease resistance in a livestock species.     

Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1

Mucolipin-1 (ML1) is a member of the transient receptor potential ion channel superfamily that is thought to function in the biogenesis of lysosomes. Mutations in ML1 result in mucolipidosis type IV, a lysosomal storage disease characterized by the intracellular accumulation of enlarged vacuolar structures containing phospholipids, sphingolipids, and mucopolysaccharides. Little is known about how ML1 trafficking or activity is regulated. Here we have examined the processing and trafficking of ML1 in a variety of cell types. We find that a significant fraction of ML1 undergoes cell type-independent cleavage within the first extracellular loop of the protein during a late step in its biosynthetic delivery. To determine the trafficking route of ML1, we systematically examined the effect of ablating adaptor protein complexes on the localization of this protein. Whereas ML1 trafficking was not apparently affected in fibroblasts from mocha mice that lack functional adaptor protein complex (AP)-3, small interfering RNA-mediated knockdown revealed a requirement for AP-1 in Golgi export of ML1. Knockdown of functional AP-2 had no effect on ML1 localization. Interestingly, cleavage of ML1 was not compromised in AP-1-deficient cells, suggesting that proteolysis occurs in a prelysosomal compartment, possibly the trans-Golgi network. Our results suggest that posttranslational processing of ML1 is more complex than previously described and that this protein is delivered to lysosomes primarily via an AP-1-dependent route that does not involve passage via the cell surface.     

Histochemical response of mice to mistletoe lectin I (ML I)

The acute toxicity of lectin ML I from the toxic drug, mistletoe, was demonstrated in previous experiments. Because the reason for this extremely high toxicity is not yet clear, mice were studied histochemically at different times after treatment with various doses of ML I, ML I A or ML I B chain separately, or recombinations of ML I A and ML I B. Various plasma membrane-associated hydrolases as well as Golgi apparatus-and endoplasmic reticulum-linked hydrolases, peroxisomal and extraperoxisomal oxidases, lysosomal hydrolases, mitochondrial dehydrogenases, the cytoskeletal proteins keratin and vimentin as well as iron, glycogen and lipids were analysed in all organs and tissues of female mice. Irrespective of the dose, a clear-cut response was only observed in the liver. After ML I treatment, glycogen disappeared completely from all hepatocytes, and this effect did not depend on the ML I-concentration and exposure time. The increase in activity of Golgi-associated thiamine pyrophosphatase in hepatocytes and of non-specific alkaline phosphatase in the sinusoidal endothelial cells depended on the applied ML I concentration and the time of treatment. Doses of 600 or 900 ng ML I/kg drastically increased the phosphatase activities. These clear-cut changes of glycogen and enzyme activities were not observed after administration of the ML I B chain alone, and less so when the mice were treated only with the ML I A chain, or were treated with a recombination of ML I A and ML I B even at concentrations higher than that of ML I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-induced conformational changes in human tearlipids hydrocarbon chains

As a first step to characterize human meibum and tear lipids, infrared spectroscopy was applied to characterize the molecular structure/conformation and packing of hydrocarbon chains. Temperature-induced phase transitions were fit to a sigmoid equation and were experimentally reproducible and were similar for multiple samples collected from the same person. No hysteresis was observed. Hydration of polar tear lipids increased their phase transition cooperativity, enthalpy and entropy. Hydrophobic interactions in meibum lipid (ML) were stronger than in tear-fluid lipids (TL), as reflected by the higher entropy and enthalpy of the gel to liquid crystalline phase transition of ML. The results of this study provide further evidence of the differences in the composition and structure of ML and TL. The conformational changes observed in the hydrocarbon chains of ML with temperature suggest that the observed therapeutic increased delivery of ML with eye lid heating could be related to the increased disorder in the packing of the hydrocarbon tails. This work also highlights the power of infrared spectroscopy to characterize molecular structure/conformation, and packing of human tear lipids and provides a basis for future studies of tear film lipid composition-structure-function relationships and lipid-protein interactions in relation to age, sex, and dry eye symptoms.     

Histochemical response of mice to mistletoe lectin I (ML I)

Summary The acute toxicity of lectin ML I from the toxic drug, mistletoe, was demonstrated in previous experiments. Because the reason for this extremely high toxicity is not yet clear, mice were studied histochemically at different times after treatment with various doses of ML I, ML I A or ML I B chain separately, or recombinations of ML I A and ML I B. Various plasma membrane-associated hydrolases as well as Golgi apparatus-and endoplasmic reticulum-linked hydrolases, peroxisomal and extraperoxisomal oxidases, lysosomal hydrolases, mitochondrial dehydrogenases, the cytoskeletal proteins keratin and vimentin as well as iron, glycogen and lipids were analysed in all organs and tissues of female mice. Irrespective of the dose, a clear-cut response was only observed in the liver. After ML I treatment, glycogen disappeared completely from all hepatocytes, and this effect did not depend on the ML I-concentration and exposure time. The increase in activity of Golgiassociated thiamine pyrophosphatase in hepatocytes and of non-specific alkaline phosphatase in the sinusoidal endothelial cells depended on the applied ML I concentration and the time of treatment. Doses of 600 or 900 ng ML I/kg drastically increased the phosphatase activities. These clear-cut changes of glycogen and enzyme activities were not observed after administration of the ML I B chain alone, and less so when the mice were treated only with the ML I A chain, or were treated with a recombination of ML I A and ML I B even at concentrations higher than that of ML I. The glycogen as well as thiamine pyrophosphatase and non-specific alkaline phosphatase responses after ML I administration were highly reproducible. None of the other investigated cell constituents responded to treatment with ML I, irrespective of the organs, tissues and cells analysed. This was also true for mice treated with ML I concentrations higher than the LD₅₀, after which the animals died with 2–3 days. In conclusion, application of ML I induces clear-cut liver cell changes, which are not observed after treatment with individual constituents of the ML I molecule either singly or in recombination. However, these changes do not explain the deaths of the animals.Supported by WTZ (Vereinbarung über wissenschaftlich-technische Zusammenarbeit: Projekt 13)     

Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.     

Application of computer to monitoring and control of fermentation process: Microbial conversion of ML-236B Na to pravastatin

An automatic feeding process for microbial hydroxylation of ML236B sodium salt (ML-236B Na; compactin) by Streptomyces carbophilus SANK 62585 was developed. The hydroxylated product, pravastatin sodium salt (pravastatin; trade name Mevalotin), is an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase (HMG-CoA reductase) used as cholesterol-lowering drug. The hydroxylation activity of S. carbophilus was induced by the addition of ML236B Na to culture broth but inhibited by high concentration of ML236B Na. In order to obtain high conversion yield, it was necessary to maintain optimum ML236B Na concentration throughout the fermentation by continuous feeding. For this purpose, we developed an on-line monitoring method, which mainly consisted of a cross-flow filtration module, high-performance liquid chromatography (HPLC) analyzer, feed pump, and microcomputer for regulation of ML236B Na concentration. An algorithm for control of ML236B Na feed rate based on feedback and feed-forward control where conversion rate after Deltat was estimated by using regression analysis of the five latest values of conversion rate. In a fed-batch culture employing this system, the concentration of ML236B Na was maintained at optimum level during the fermentation and the productivity of pravastatin was increased threefold over that obtained in manual control culture. (c) 1993 John Wiley & Sons, Inc.     

Pulmonary absorption of liposomal levonorgestrel

The purpose of these studies was to achieve desired bioavailability after pulmonary administration of Levonorgestrel (LN) and to provide prolonged effective concentration of the drug in plasma and to reduce reported side effects of orally administered drug. The plain drug suspension, physical mixture (plain drug with liposomal constituents), and drug-encapsulated liposomes containing 10 μg of drug were instilled intratracheally in rats. Similarly, 10-μg drug suspension (LO) was administered orally. The blood samples were withdrawn at specific time intervals and were subjected to LN analysis by spectrofluorimetric technique. The plasma drug concentration data of both the treatments were plotted, and pharmacokinetics data were calculated and compared with that of oral administration. Percentage relative bioavailability (F^*) of 97.6% 98.6%, and 109.9% were observed after pulmonary administration of plain drug formulation (LP1), physical mixture (plain drug along with constituents of liposomes [LP2], and liposomal (LP3) formulations of the drug, respectively. Following oral administration, C_max of 14.4±0.6 ng/mL was observed at 2.1±0.2 hours followed by subtherapeutic concentration beyond 30±0.2 hours, while after pulmonary administration of LP1, LP2, and LP3 formulations, C_max of 4.4±0.4 ng/mL, 4.2±0.5 ng/mL, and 4.4±0.6 ng/ML were observed at 6.0±0.2 hours, 7.0±0.2 hours, and 6.8±0.2 hours, respectively, followed by maintenance of effective plasma drug concentration up to 60±2 hours. These studies demonstrate superiority of pulmonary drug delivery with regards to maintenance of effective therapeutic concentration of the LN in the plasma over a period of 6 to 60 hours. Hence, the pulmonary delivery is expected to reduce frequency of dosing and systemic side effects associated with oral administration of LN.     

The role of multiscale computational approaches for rational design of conventional and nanoparticle oral drug delivery systems

Multiscale computational modeling of drug delivery systems (DDS) is poised to provide predictive capabilities for the rational design of targeted drug delivery systems, including multi-functional nanoparticles. Realistic, mechanistic models can provide a framework for understanding the fundamental physico-chemical interactions between drug, delivery system, and patient. Multiscale computational modeling, however, is in its infancy even for conventional drug delivery. The wide range of emerging nanotechnology systems for targeted delivery further increases the need for reliable in silico predictions. This review will present existing computational approaches at different scales in the design of traditional oral drug delivery systems. Subsequently, a multiscale framework for integrating continuum, stochastic, and computational chemistry models will be proposed and a case study will be presented for conventional DDS. The extension of this framework to emerging nanotechnology delivery systems will be discussed along with future directions. While oral delivery is the focus of the review, the outlined computational approaches can be applied to other drug delivery systems as well.     

Endocytosis in cellular uptake of drug delivery vectors: Molecular aspects in drug development

Drug delivery vectors are widely applied to increase drug efficacy while reducing the side effects and potential toxicity of a drug. They allow for patient-tailored therapy, dose titration, and therapeutic drug monitoring. A major part of drug delivery systems makes use of large nanocarriers: liposomes or virus-like particles (VLPs). These systems allow for a relatively large amount of cargo with good stability of vectors, and they offer multiple options for targeting vectors in vivo. Here we discuss endocytic pathways that are available for drug delivery by large nanocarriers. We focus on molecular aspects of the process, including an overview of potential molecular targets for studies of drug delivery vectors and for future solutions allowing targeted drug delivery.     

The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-gamma-aspartate to cells in vitro

We have studied the liposome-mediated delivery of methotrexate-gamma-aspartate to five cell lines. The sensitivity of the cells to encapsulated drug varies widely in accordance with their ability to take up the liposomes. CV1-P cells can be 150-times more sensitive to encapsulated methotrexate-gamma-aspartate than to free drug, while AKR/J SL2 cells are only twice as sensitive to the encapsulated drug. Negatively-charged liposomes are much more efficient for delivery than are neutral liposomes, and cholesterol is an essential component of the liposome membrane for optimal drug delivery. The optimal liposome size for drug delivery is 0.1 micron, although the amount of cell-associated lipid is the same for all liposome sizes. The effect of the encapsulated drug is inhibited by NH4Cl, suggesting an endocytic mechanism for delivery. The potency of the encapsulated drug is not affected by wide variations in the drug: lipid ratio.     

Organic–Inorganic Composites for Bone Drug Delivery

This review paper attempts to provide an overview in the fabrication and application of organic–inorganic based composites in the field of local drug delivery for bone. The concept of local drug delivery exists for a few decades. However, local drug delivery in bone and specially application of composites for delivery of drugs to bone is an area for potential research interest in the recent time. The advantages attained by an organic–inorganic composite when compared to its individual components include their ability to release drug, adopting to the natural environment and supporting local area until complete bone regeneration, which make them carriers of interest for local drug delivery for bone.