Application of Cryopreserved Human Intestinal Mucosa and Cryopreserved Human Enterocytes in the Evaluation of Herb-Drug Interactions: Evaluation of CYP3A Inhibitory Potential of Grapefruit Juice and Commercial Formulations of Twenty-Nine Herbal Supplements.

Commercial formulations of 29 commonly used herbal supplements (HSs) and grapefruit juice were evaluated for drug interaction potential via quantification of their CYP3A inhibitory potential in two in vitro experimental models of human small intestine, cryopreserved human intestinal mucosa (CHIM), and cryopreserved human enterocytes (CHEs). Two CYP3A substrates were used-in the studies with CHIM, CYP3A activity was quantified via liquid chromatography tandem mass spectrometry quantification of midazolam 1'-hydroxylation, whereas in CHE, luciferin-IPA metabolism to luciferin was quantified by luminescence. Upon treatment of CHIM with the estimated lumen concentration of the HS upon each oral administration (manufacturers' recommended dosage dissolved in 200 ml of culture medium), >80% CYP3A inhibition was observed for green tea extract, St. John's wort, valerian root, horehound, and grapefruit juice. Less than 50% inhibition was observed for fenugreek, aloe vera, guarana, soy isoflavone, maca, echinacea, spirulina, evening primrose, milk thistle, cranberry, red yeast rice, rhodiola, ginkgo biloba, turmeric, curcumin, white kidney bean, garlic, cinnamon, saw palmetto berries, panax ginseng, black elderberry, wheat grass juice, flaxseed oil, black cohosh, and ginger root. The results were confirmed in a a dose-response study with HSs obtained from three suppliers for the four inhibitory HSs (green tea extract, horehound, St. John's wort, valerian root) and three representative noninhibitory HSs (black cohosh, black elderberry, echinacea). Similar results were obtained with the inhibitory HSs in CHE. The results illustrate that CHIM and CHE represent physiologically relevant in vitro experimental models for the evaluation of drug interaction potential of herbal supplements. Based on the results, green tea extract, horehound, St. John's wort, and valerian root may cause drug interactions with orally administered drugs that are CYP3A substrates, as was observed for grapefruit juice. SIGNIFICANCE STATEMENT: In vitro evaluation of 29 popular herbal supplements in cryopreserved human intestinal mucosa identified green tea extract, horehound, St. John's wort, and valerian root to have CYP3A inhibitory potential similar to that for grapefruit juice, suggesting their potential to have clinically significant pharmacokinetic interaction with orally administered drugs that are CYP3A substrates. The results suggest that cryopreserved human intestinal mucosa can be used for in vitro evaluation of drug interactions involving enteric drug metabolism.

A 13-week subchronic toxicity study of vanillin propylene glycol acetal in F344 rats.

Vanillin propylene glycol acetal (VPGA) has been used as a flavoring agent. Here, we performed a 13-week subchronic toxicity study of VPGA in F344 rats with oral administration by gavage at doses of 0, 100, 300, and 1000 mg/kg body weight (BW)/day. In the 1000 mg/kg BW group, loss of vigorous activity and listlessness immediately after administration were observed for both sexes throughout the experimental period. Reduction of body weight gain was noted in both sexes at 1000 mg/kg BW. Serum biochemistry demonstrated significant increases in total protein, albumin, total cholesterol, calcium, inorganic phosphorus, and γ-glutamyl transpeptidase in both sexes at 1000 mg/kg BW and increases in the albumin/globulin ratio and urea nitrogen in the male 1000 mg/kg BW group. A significant increase in relative liver weight was detected in both sexes at 1000 mg/kg BW. Histopathologically, centrilobular hepatocellular hypertrophy in the liver was observed in both sexes at 1000 mg/kg BW. In addition, the incidence of fatty changes in hepatocytes in the male 1000 mg/kg BW group was decreased compared with that in the control. Based on these results, the no-observed-adverse-effect level for VPGA was evaluated to be 300 mg/kg BW/day for both sexes in the current study.

Malaria-infected mice live until at least day 30 after a new artemisinin-derived thioacetal thiocarbonate combined with mefloquine are administered together in a single, low, oral dose.

In only three steps and in 21-67% overall yields from the natural trioxane artemisinin, a series of 21 new trioxane C-10 thioacetals was prepared. Upon receiving a single oral dose of only 6 mg/kg of the monomeric trioxane 12c combined with 18 mg/kg of mefloquine hydrochloride, Plasmodium berghei-infected mice survived on average 29.8 days after infection. Two of the four mice in this group had no parasites detectable in their blood on day 30 after infection, and they behaved normally and appeared healthy. One of the mice had 11% blood parasitemia on day 30, and one mouse in this group died on day 29. Of high medicinal importance, the efficacy of this ACT chemotherapy is much better than (almost double) the efficacy under the same conditions using as a positive control the popular trioxane drug artemether plus mefloquine hydrochloride (average survival time of only 16.5 days).

Antihyperglycaemic activity of 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal in diabetic mice.

We have recently generated lipophilic D-xylose derivatives that increase the rate of glucose uptake in cultured skeletal muscle cells in an AMP-activated protein kinase (AMPK)-dependent manner. The derivative 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal (EH-36) stimulated the rate of glucose transport by increasing the abundance of glucose transporter-4 in the plasma membrane of cultured myotubes. The present study aimed at investigating potential antihyperglycaemic effects of EH-36 in animal models of diabetes. Two animal models were treated subcutaneously with EH-36: streptozotocin-induced diabetes in C57BL/6 mice (a model of insulin-deficient type 1 diabetes), and spontaneously diabetic KKAy mice (Kuo Kondo rats carrying the A(y) yellow obese gene; insulin-resistant type 2 diabetes). The in vivo biodistribution of glucose in control and treated mice was followed with the glucose analogue 2-deoxy-2-[(18) F]-D-glucose; the rate of glucose uptake in excised soleus muscles was measured with [(3) H]-2-deoxy-D-glucose. Pharmacokinetic parameters were determined by non-compartmental analysis of the in vivo data. The effective blood EH-36 concentration in treated animals was 2 µM. It reduced significantly the blood glucose levels in both types of diabetic mice and also corrected the typical compensatory hyperinsulinaemia of KKAy mice. EH-36 markedly increased glucose transport in vivo into skeletal muscle and heart, but not to adipose tissue. This stimulatory effect was mediated by Thr(172) -phosphorylation in AMPK. Biochemical tests in treated animals and acute toxicological examinations showed that EH-36 was well tolerated and not toxic to the mice. These findings indicate that EH-36 is a promising prototype molecule for the development of novel antidiabetic drugs.

Methylglyoxal and other carbohydrate metabolites induce lanthanum-sensitive Ca2+ transients and inhibit growth in E. coli.

The results here are the first demonstration of a family of carbohydrate fermentation products opening Ca2+ channels in bacteria. Methylglyoxal, acetoin (acetyl methyl carbinol), diacetyl (2,3 butane dione), and butane 2,3 diol induced Ca2+ transients in Escherichia coli, monitored by aequorin, apparently by opening Ca2+ channels. Methylglyoxal was most potent (K(1/2) = 1 mM, 50 mM for butane 2,3 diol). Ca2+ transients depended on external Ca2+ (0.1-10 mM), and were blocked by La3+ (5 mM). The metabolites affected growth, methylglyoxal being most potent, blocking growth completely up to 5 h without killing the cells. But there was no affect on the number of viable cells after 24 h. These results were consistent with carbohydrate products activating a La3+-sensitive Ca2+ channel, rises in cytosolic Ca2+ possibly protecting against certain toxins. They have important implications in bacterial-host cell signalling, and where numbers of different bacteria compete for the same substrates, e.g., the gut in lactose and food intolerance.

Acetals as pH-sensitive linkages for drug delivery.

pH-Sensitive linkages designed to undergo hydrolysis at mildly acidic pH can trigger the release of therapeutics selectively at targets such as tumor and inflammatory tissues and in the endosomes and lysosomes of cells. Acetals have the potential to be used as linkages for a range of alcohol functionalities, and, by altering their chemical structure, it is possible to tune their hydrolysis rate. The syntheses of four conjugates of model drug molecules with PEO using acetals of varying chemical structure are described herein. Primary and secondary alcohols, as well as syn-1,2-diols, were incorporated in the conjugates. The hydrolysis kinetics were investigated by HPLC, and the conjugates had half-lives ranging from less than 1 min to several days at pH 5.0, with slower hydrolysis at pH 7.4 in all cases. These acetal linkages are therefore promising for use in a variety of drug delivery applications ranging from polymer-drug conjugates to pH-sensitive micelles and nanoparticulate systems.

Substance P receptor antagonist I: conversion of phosphoramidate prodrug after i.v. administration to rats and dogs.

A water-soluble phosphoramidate prodrug (L-758,298, compound I) of the potent and selective human Substance P receptor antagonist L-754, 030 (compound II) is under development as an i.v. drug for treatment of emesis, migraine, and chronic pain. Compound I undergoes hydrolysis readily to II under acidic conditions. In the studies reported herein, we investigated the stability of I in blood and hepatic subcellular fractions from rats, dogs, and humans as well as the conversion of I to II in rats and dogs after i.v. dosing. Compound I was converted to II rapidly in rat blood but was stable in dog and human blood. However, the conversion was rapid in liver microsomes prepared from dogs and humans. As expected from the results of in vitro studies, the in vivo conversion of I to II was rapid after i.v. dosing of I to rats and dogs. The relative extent of exposure of II after i.v. dosing of I was estimated by comparing the dose-adjusted area under the plasma concentration versus time curve values of II after i.v. dosing of I with those after i.v. dosing of II. In rats, the extent of exposure was estimated to be approximately 90 and approximately 100% at 1 and 8 mg/kg, respectively; in dogs, that was approximately 59% at 0.5 mg/kg. A nonproportional increase in the area under the concentration versus time curve value of II with dose was observed after i.v. administration of I in dogs from 0.5 to 32 mg/kg, suggesting that the elimination of II might have been saturated at higher doses.

Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist.

Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.

Biodegradable polymers in controlled drug delivery.

Erosion mechanisms are divided into three types and drug release within each type is described. Type I erosion involves hydrolysis of hydrogels and these are useful in the controlled release of macromolecules entangled within their network structure. Type II erosion involves solubilization of water-insoluble polymers by reactions involving groups pendant from the polymer backbone. Of particular interest are polymers that solubilize by ionization of carboxylic acid groups, and the utilization of those systems is described. Type III erosion involves cleavage of hydrolytically labile bonds within the polymer backbone and four distinct polymer systems within this category are under development. One system involves the diffusion of drugs from a reservoir through a bioerodible membrane, another system utilizes microcapsules, a third system utilizes monolithic devices, and the fourth system utilizes drugs chemically bound to a bioerodible polymer.

PIP: Controlled-release methodologies can be classified on the basis of the mechanism that controls the release of the active agent from the delivery device diffusion, osmosis, or polymer erosion. This paper presents an extensive review of the research on erosional devices. The various polymer erosion mechanisms are of 3 basic types. Type I erosion refers to water-soluble polymers that have been insolubilized by covalent cross-links and that solubilize as the cross-links (type IA) or backbone (type IB) undergo a hydrolytic cleavage. In type II erosion, polymers that are initially water insoluble are solubilized by hydrolysis, ionization, or pronation of a pendant group. In type III erosion, hydrophobic polymers are converted to small water-soluble molecules by backbone cleavage. The choice of a particular erosion mechanism is dictated by the specific application. In addition, there are 2 mechanisms of polymer release from bioerodible polymers: one approach involves surrounding the drug core with a rate-controlling bioerodible membrane, while the other involves dispersing the drug within a polymer to form a bioerodible monolithic device. The use of biodegradable systems for the sustained release of fertility-regulating agents is based on type III erosion. Polymer erosion tends to lead drug release, and there is some indication that drug release from the implant is controlled by rate of solubilization of the highly water-insoluble steroid.