Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-toward a consensus.
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Animals↗
Biomedical subjects
S M Huang
Publications and source records attributed to S M Huang.
Cannabinoids and pain.
Cannabinoids have been used to treat pain for many centuries. However, only during the past several decades have rigorous scientific methods been applied to understand the mechanisms of cannabinoid action. Cannabinoid receptors were discovered in the late 1980s and have been found to mediate the effects of cannabinoids on the nervous system. Several endocannabinoids were subsequently identified. Many studies of cannabinoid analgesia in animals during the past century showed that cannabinoids block all types of pain studied. These effects were found to be due to the suppression of spinal and thalamic nociceptive neurons, independent of any actions on the motor systems. Spinal, supraspinal and peripheral sites of cannabinoid analgesia have been identified. Endocannabinoids are released upon electrical stimulation of the periaqueductal gray, and in response to inflammation in the extremities. These observations and others thus suggest that a natural function of cannabinoid receptors and their endogenous ligands is to regulate pain sensitivity. The therapeutic potential of cannabinoids remains an important topic for future investigations, with previous work suggesting utility in clinical studies of cancer and surgical pain. New modes of delivery and/or new compounds lacking the psychotropic properties of the standard cannabinoid ligands offer promise for cannabinoid therapeutics for pain.
Analgesia↗
Prophylactic total thyroidectomy in an 8-year-old girl with multiple endocrine neoplasia type 2A.
We report a genetic carrier of multiple endocrine neoplasia type 2A (MEN 2A) who underwent prophylactic total thyroidectomy. The asymptomatic carrier of MEN 2A, an 8-year-old Taiwanese girl, was admitted for early thyroidectomy. Preoperative basal plasma concentrations of calcitonin and intact parathyroid hormone, and urine vanillylmandelic acid excretion, were normal. Ultrasonography of the thyroid was also normal. Pathology did not reveal any gross lesion but C-cell hyperplasia of the thyroid gland was found microscopically. Prophylactic total thyroidectomy is encouraged and is justified during the first decade of life for MEN 2A carriers.
Child↗
The effects of St John's wort (Hypericum perforatum) on human cytochrome P450 activity.
BACKGROUND: St John's wort (Hypericum perforatum) is a popular over-the-counter dietary supplement and herbal remedy that has been implicated in drug interactions with substrates of several cytochrome P450 (CYP) isozymes. The effect of St John's wort on CYP activity in vivo was examined with a probe drug cocktail. METHODS: Twelve healthy subjects (5 female, 7 male) completed this 3-period, open-label, fixed schedule study. Tolbutamide (CYP2C9), caffeine (CYP1A2), dextromethorphan (CYP2D6), oral midazolam (intestinal wall and hepatic CYP3A), and intravenous midazolam (hepatic CYP3A) were administered before, with short-term St John's wort dosing (900 mg), and after 2 weeks of intake (300 mg 3 times a day) to determine CYP activities. RESULTS: Short-term administration of St John's wort had no effect on CYP activities. Long-term St John's wort administration caused a significant (P <.05) increase in oral clearance of midazolam from 121.8 +/- 70.7 to 254.5 +/- 127.8 and a corresponding significant decline in oral bioavailability from 0.28 +/- 0.15 to 0.17 +/- 0.06. In contrast to the >50% decrease in the area under the plasma concentration-time curve (AUC) when midazolam was administered orally, long-term St John's wort administration caused a 20% decrease in AUC when midazolam was given intravenously. There was no change in CYP1A2, CYP2C9, or CYP2D6 activities as a result of St John's wort administration. CONCLUSION: Long-term St John's wort administration resulted in a significant and selective induction of CYP3A activity in the intestinal wall. St John's wort did not alter the CYP2C9, CYP1A2, or CYP2D6 activities. Reduced therapeutic efficacy of drugs metabolized by CYP3A should be anticipated during long-term administration of St John's wort.
Administration, Oral↗
Monoclonal antibody blockade of the epidermal growth factor receptor in cancer therapy.
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Animals↗
Synergistic, p160 coactivator-dependent enhancement of estrogen receptor function by CARM1 and p300.
Members of the p160 coactivator family (steroid receptor coactivator-1 (SRC-1), glucocorticoid receptor interacting protein 1 (GRIP1), and activator of thyroid and retinoic acid receptors (ACTR)) mediate transcriptional activation by nuclear receptors. After being recruited to the promoter by nuclear receptors, the p160 coactivator transmits the activating signal via two C-terminal activation domains, AD1 and AD2. AD1 is a binding site for the related coactivators cAMP-response element binding protein binding protein (CBP) and p300, whereas AD2 binds to another coactivator, coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase. The current study explored the cooperative functional and mechanistic relationships among GRIP1, CARM1, and p300 in transient transfection assays, where they enhanced the ability of the estrogen receptor (ER) to activate transcription of a reporter gene. The coactivator functions of p300 and CARM1 depended on the co-expression of GRIP1. Simultaneous co-expression of all three coactivators caused a synergistic enhancement of ER function. Deletion of the AD1 domain of GRIP1 abolished the ability of p300 to potentiate ER activity but had no effect on CARM1-mediated stimulation. In contrast, when the AD2 domain of GRIP1 was deleted, p300 still stimulated ER function through the mutant GRIP1, but CARM1 failed to do so. Thus, both binding of p300 to AD1 and binding of CARM1 to AD2 are required for their respective coactivator functions and for their synergy. Furthermore, CARM1 and p300 function independently through different activating domains of GRIP1, and their synergy suggests that they enhance transcription by different, complementary mechanisms.
Cell Line↗
Prognostic value of vascular endothelial growth factor expression in colorectal cancer patients.
Solid tumours require neovascularisation for growth and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be an important regulator of tumour angiogenesis. To examine the relevance of VEGF in the neoplastic transformation of human colon, we analysed protein expression in a total 30 polyps and 145 colorectal carcinomas by immunohistochemistry. All adenoma specimens, regardless of histological differentiation, and normal colonic mucosa did not express VEGF. Amongst 90 patients with non-metastatic colorectal cancer, VEGF expression was observed in 43 (48%) cases, whilst 29 of the 55 patients (53%) with metastases expressed the angiogenic factor. Both the proportion and intensity of VEGF expression were positively associated with the progression of colon carcinogenesis. Tumours with the highest VEGF expression tended to correlate with patients' survival, although VEGF expression did not emerge as an independent risk factor in a multivariate analysis. After exclusion of the patients with distant metastases, both univariate and multivariate analysis did not indicate any prognostic value for the tissues with the highest VEGF expression. Our results suggest that VEGF may play a role in the progression of colon cancer, although evaluation of this angiogenic phenotype did not provide additional prognostic information compared with that obtained from Dukes' staging of the tumours.
Adenoma↗
Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes.
1. Troglitazone was the first thiazolidinedione approved for clinical use in the treatment of non-insulin-dependent diabetes mellitus. During clinical investigations of drug-drug interactions with therapeutics (terfenadine and cyclosporine) known to be metabolized by CYP3A4, pharmacokinetic interactions were noted upon troglitazone multiple-dose treatments. The nature of the interactions suggested induction of CYP3A enzymes. 2. Primary cultures of human hepatocytes were used to investigate the induction potential of troglitazone with respect to CYP3A4, CYP2B6 and CYP1A1/2. In human hepatocytes, troglitazone induced both immunoreactive CYP3A4 protein and testosterone 6beta-hydroxylase activity in a dose-dependent fashion (EC50 = 5-10 microM), accompanied by an increase in CYP3A4 mRNA. The capacity of troglitazone to induce CYP3A4 was between that of rifampin (EC50 = 0.8 microM) and dexamethasone (40-50 microM). Troglitazone increased CYP2B6 immunoreactive protein but did not significantly effect CYP1A1/2 activity, immunoreactive protein or mRNA. 3. Troglitazone produced significant increases in CYP3A message, protein and activity in primary rat hepatocytes, a slight increase in CYP2B1/2 activity and no change in CYP1A1/2 message or activity. 4. These results provide evidence that troglitazone can induce CYP3A and CYP2B enzymes while apparently not altering CYP1A. This provides a rationale for the clinically observed interactions of troglitazone with selected CYP3A4 substrates.
Animals↗
Mouse Zac1, a transcriptional coactivator and repressor for nuclear receptors.
Transcriptional activation by nuclear hormone receptors is mediated by the 160-kDa family of nuclear receptor coactivators. These coactivators associate with DNA-bound nuclear receptors and transmit activating signals to the transcription machinery through two activation domains. In screening for mammalian proteins that bind the C-terminal activation domain of the nuclear receptor coactivator GRIP1, we identified a new variant of mouse Zac1 which we call mZac1b. Zac1 was previously discovered as a putative transcriptional activator involved in regulation of apoptosis and the cell cycle. In yeast two-hybrid assays and in vitro, mZac1b bound to GRIP1, to CREB-binding protein (CBP) and p300 (which are coactivators for nuclear receptors and other transcriptional activators), and to nuclear receptors themselves in a hormone-independent manner. In transient-transfection assays mZac1b exhibited a transcriptional activation activity when fused with the Gal4 DNA binding domain, and it enhanced transcriptional activation by the Gal4 DNA binding domain fused to GRIP1 or CBP fragments. More importantly, mZac1b was a powerful coactivator for the hormone-dependent activity of nuclear receptors, including androgen, estrogen, glucocorticoid, and thyroid hormone receptors. However, with some reporter genes and in some cell lines mZac1b acted as a repressor rather than a coactivator of nuclear receptor activity. Thus, mZac1b can interact with nuclear receptors and their coactivators and play both positive and negative roles in regulating nuclear receptor function.
Animals↗
Clinical pharmacology studies in patients with renal impairment: past experience and regulatory perspectives.
The objective of this report is to provide a regulatory perspective on the quality of pharmacokinetic studies in renal impairment (RI) studies submitted in support of new drug applications (NDAs) or supplements to NDAs (sNDAs) submitted to the Food and Drug Administration (FDA). Fifty-one NDA and 20 sNDA submissions reviewed between 1996 and 1997 by the Office of Clinical Pharmacology and Biopharmaceutics were evaluated for the following: (1) whether an RI study was conducted; (2) contribution of the renal clearance to the overall clearance in subjects without renal impairment; (3) degree of plasma protein binding (%PB) in subjects without renal impairment; (4) dose proportionality of single and multiple doses; (5) study design, including dosing regimen; (6) definition of renal impairment; (7) stratification of renal functions; (8) number of subjects/group; (9) data analysis and interpretation; and (10) impact on labeling. Results of the analysis indicated that 67% of the NDAs and 30% of supplemental NDAs contained RI-studies (34/51 for NDAs and 6/20 for sNDAs). No obvious differences in the pharmacokinetic characteristics (e.g., percentage excreted unchanged in urine and %PB) were observed between drugs for which RI studies were conducted versus those not conducted. Most studies conducted were designed as single dose (70%). Seventy-five percent of the studies used doses within the therapeutic dosage range of the drug. The measured 24-hour creatinine clearance was most often used to assess the renal function. Stratification of renal function ranged from one to five groups, with 6 to 8 subjects enrolled per group. In most studies conducted (38/40), data were analyzed by point estimate using ANOVA. Results of RI studies were adequately reflected in the labeling. The survey reveals that RI study design can be improved for regulatory review purposes. In part based on this analysis, the FDA has prepared a guidance that provides recommendations on the design, analysis, and impact on dosing and labeling for RI studies to include recommendations on when RI studies do not need to be performed. The guidance proposes an equivalence approach with confidence intervals, as opposed to a point estimate approach, to assess the impact of RI on systemic exposure measures.
Attitude of Health Personnel↗
Modulation of molecular targets to enhance radiation.
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Animals↗
Modulation of radiation response after epidermal growth factor receptor blockade in squamous cell carcinomas: inhibition of damage repair, cell cycle kinetics, and tumor angiogenesis.
We have recently demonstrated that molecular blockade of the epidermal growth factor receptor with the anti-epidermal growth factor receptor (EGFR) monoclonal antibody C225 enhances the in vitro radiosensitivity of human squamous cell carcinomas (SCCs) derived from the head and neck. In the present study, we further investigated the capacity of C225 to modulate the in vitro and in vivo radiation response of human SCC tumor cells and xenografts, and we examined several potential mechanisms that may contribute to the enhanced radiation response induced by C225. Tumor xenograft studies demonstrated complete regression of both newly established (20 mm3) and well-established (100 mm3) SCC tumors over a 55-100 day follow-up period in athymic mice treated with the combination of C225 (i.p. injection) and radiation. Cell cycle analysis via flow cytometry confirmed that combined treatment with C225 and radiation induced an accumulation of cells in the more radiosensitive cell cycle phases (G1, G2-M) with concurrent reduction in the proportion of cells in the more radioresistant S phase. Results from sublethal damage repair and potentially lethal damage repair analyses in cultured SCC cells demonstrated a strong inhibitory effect of C225 on postradiation damage repair. Further, exposure of SCC cells to C225 induced a redistribution of DNA-dependent protein kinase from the nucleus to the cytosol, suggesting one potential mechanism whereby C225 may influence the cellular response to radiation. Immunohistochemical analysis of SCC tumor xenografts after systemic administration of C225 demonstrated inhibition of the in vivo expression of tumor angiogenesis markers, including vascular endothelial growth factor and Factor VIII. Taken together, the collective data suggest that the profound in vivo antitumor activity identified in the xenograft setting when C225 is combined with radiation derives from more than simply the antiproliferative and cell cycle effects of EGFR system inhibition. In addition to antiproliferative growth inhibition, EGFR blockade with C225 appears to influence the capacity of human SCCs to effect DNA repair after exposure to radiation, and to express classic markers of tumor angiogenesis.
Animals↗
Effects of VEGF121 and/or VEGF165 gene transfection on collateral circulation development.
BACKGROUND AND PURPOSE: Angiogenesis is regulated by various factors, including vascular endothelial growth factor (VEGF). Five isoforms of VEGF have been discovered: VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. The teleologic basis for the various VEGF isoforms remains unclear, but different VEGF isoforms may mediate distinct endothelial cell functions such as angiogenesis, vascular permeability, and differentiation. We sought to determine the effects of various VEGF isoforms on angiogenesis under ischemic conditions in rabbits. METHODS: The effects of VEGF121 and/or VEGF165 gene transfection on collateral circulation development in ischemic rabbit hindlimb muscles were investigated by using naked plasmids encoding VEGF121 or VEGF165 (pVEGF121 or pVEGF165), either individually or in combination. pCMV beta was used as the control plasmid. The femoral artery on one side of New Zealand White rabbits was ligated. Ten days later, the ischemic muscles received direct intramuscular injection of pVEGF121 (500 micrograms), pVEGF165 (500 micrograms), or pVEGF121 (250 micrograms) + pVEGF165 (250 micrograms) in experimental groups, while pCMV beta (500 micrograms) was used in the control group. Therapeutic effects were evaluated 30 days later by anatomic and physiologic analysis. RESULTS: Internal iliac angiography showed strong development of collateral circulation in all of the pVEGF-treated groups. In contrast, collateral arteries developed weakly in the control group. Combination treatment with both pVEGF121 and pVEGF165 did not result in additional improvement compared with pVEGF121 or pVEGF165 treatment alone (angiographic scores: pVEGF121 = 0.85 +/- 0.10; pVEGF165 = 0.81 +/- 0.11; pVEGF121 + pVEGF165 = 0.83 +/- 0.09; control = 0.53 +/- 0.09; p < 0.01). A favorable response in the development of circulation at the capillary level with pVEGF121 and/or pVEGF165 versus pCMV beta was also found. Blood pressure measurement and regional blood flow measurement using colored microspheres revealed similar results. CONCLUSIONS: Our results show that direct intramuscular injection of naked DNA encoding VEGF121 or VEGF165, individually or in combination, is an effective method for gene transfer in an animal model of ischemic limbs and results in augmented collateral vascular development and tissue perfusion.
Angiography↗
In vitro dissolution of cholesterol and brown pigmented gallstones: a comparison of MTBE, DMSO and BA-EDTA.
BACKGROUND: Gallstones are a common problem in Taiwan and surgical removal remains the essential treatment. Successful dissolution of the stones with chemical solutions and then removal by endoscopic or percutaneous methods have previously been reported. We designed this study to find the ideal agent for dissolving gallstones. METHODS: Twelve chemical solutions with dimethylsulfoxide (DMSO), methyl tert-butyl ether (MTBE) and ethylenediamine tetra-acetic acid (EDTA) in different mixtures were tested to investigate their ability to dissolve gallstones in vitro. The dissolution of stones was performed at 37 degrees C and each procedure was repeated five to seven times. RESULTS: The solvent containing DMSO/MTBE (1/1) had a higher dissolving capacity for cholesterol stones, with solubility reaching 96.8% after 6 hours. The solution containing DMSO/MTBE (7/3) had the maximal solubility for calcium bilirubinate stones, with solubility reaching 22.9% after 6 hours. Also, we found that the intact stones of calcium bilirubinate became fragmented after treatment with the DMSO/MTBE solution without stirring. CONCLUSIONS: The DMSO, MTBE and EDTA agents that we used effectively dissolved gallstones, especially cholesterol stones, in vitro. Further in vivo studies are necessary to confirm the efficacy and safety of these solvents before clinical application.
Bilirubin↗
Pain modulation by release of the endogenous cannabinoid anandamide.
Synthetic cannabinoids produce behavioral analgesia and suppress pain neurotransmission, raising the possibility that endogenous cannabinoids serve naturally to modulate pain. Here, the development of a sensitive method for measuring cannabinoids by atmospheric pressure-chemical ionization mass spectrometry permitted measurement of the release of the endogenous cannabinoid anandamide in the periaqueductal gray (PAG) by in vivo microdialysis in the rat. Electrical stimulation of the dorsal and lateral PAG produced CB1 cannabinoid receptor-mediated analgesia accompanied by a marked increase in the release of anandamide in the PAG, suggesting that endogenous anandamide mediates the behavioral analgesia. Furthermore, pain triggered by subcutaneous injections of the chemical irritant formalin substantially increased the release of anandamide in the PAG. These findings indicate that the endogenous cannabinoid anandamide plays an important role in a cannabinergic pain-suppression system existing within the dorsal and lateral PAG. The existence of a cannabinergic pain-modulatory system may have relevance for the treatment of pain, particularly in instances where opiates are ineffective.
Analgesia↗
The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300.
The co-activators CBP and p300 are important for normal cell differentiation and cell cycle progression and are the targets for viral proteins that dysregulate these cellular processes. We show here that the E6 protein from the oncogenic human papillomavirus type 16 (HPV-16) binds to three regions (C/H1, C/H3 and the C-terminus) of both CBP and p300. The interaction of E6 with CBP/p300 was direct and independent of proteins known to bind the co-activators, such as p53. The E6 protein from low-risk HPV type 6 did not interact with C/H3 or the C-terminus but associated with the C/H1 domain at 50% of the level of HPV-16. HPV-16 E6 inhibited the intrinsic transcriptional activity of CBP/p300 and decreased the ability of p300 to activate p53- and NF-kappaB-responsive promoter elements. Interestingly, some mutations in HPV-16 E6 abrogated C/H3-E6 interactions, but did not alter the ability of E6 to associate with the C/H1 domain, suggesting that these modified proteins could be used to delineate the functional significance of the C/H1 and C/H3 domains of CBP/p300.
Animals↗
Calcium and phosphatidylserine stimulate the self-association of conventional protein kinase C isoforms.
Conflicting evidence exists as to whether "conventional" protein kinase C isoforms (cPKCs) function as monomers or oligomers. In this report, we demonstrate that purified cPKC isoforms can be rapidly cross-linked by the sulfhydryl-selective cross-linker bis(maleimido)hexane, but only in the presence of both Ca(2+) and phosphatidylserine; cross-linking was minimal in the presence of either of these activators alone. In addition, cross-linking of these cPKCs did not require Mg(2+) or ATP. Among the various phospholipids tested, phosphatidylserine was found to be the most effective in the promotion of cPKC self-association and for the stimulation of protein kinase activity toward the exogenous substrate histone. Phosphatidic acid and phosphatidylinositol were less effective in this regard, whereas phosphatidylcholine exhibited little ability to induce cPKC self-association or to stimulate kinase activity. An examination of the mechanism by which the cPKC isoforms self-associate in the presence of phospholipid/Ca(2+) revealed that this process occurred independently of phospholipid aggregation. Moreover, self-association was not inhibited by saturating the enzyme active site with a peptide substrate, suggesting that self-association is distinct from an enzyme-substrate interaction. Isoform-specific antibodies revealed that all cPKC isoforms (alpha, beta, and gamma) self-associate and that, in a mixture of cPKC isoforms, PKC-alpha forms primarily alpha-alpha homodimers. Besides cPKC interactions detected with purified enzyme, PKC-alpha also appeared capable of self-association in murine B82L fibroblasts that were treated with calcium ionophore, phorbol ester, or epidermal growth factor but not in untreated cells. Collectively, these data indicate that self-association occurs in parallel with cPKC activation, that self-association is not mediated by the substrate binding site, and, at least in the case of PKC-alpha, that the formation of isoform homodimers predominates.
Animals↗
Regulation of transcription by a protein methyltransferase.
The p160 family of coactivators, SRC-1, GRIP1/TIF2, and p/CIP, mediate transcriptional activation by nuclear hormone receptors. Coactivator-associated arginine methyltransferase 1 (CARM1), a previously unidentified protein that binds to the carboxyl-terminal region of p160 coactivators, enhanced transcriptional activation by nuclear receptors, but only when GRIP1 or SRC-1a was coexpressed. Thus, CARM1 functions as a secondary coactivator through its association with p160 coactivators. CARM1 can methylate histone H3 in vitro, and a mutation in the putative S-adenosylmethionine binding domain of CARM1 substantially reduced both methyltransferase and coactivator activities. Thus, coactivator-mediated methylation of proteins in the transcription machinery may contribute to transcriptional regulation.
Amino Acid Sequence↗