Mohammad K. Ashfaq

Indolizidine, antiinfective and antiparasitic compounds from Prosopis glandulosa var. glandulosa.

A new potent antiinfective and antiparasitic 2,3-dihydro-1H-indolizinium chloride (1) was isolated from Prosopis glandulosa var. glandulosa. Three additional new (2-4) and one known (5) indolizidines were also isolated, and the dihydrochloride salts of 1-3 (compounds 6, 7, and 8) were prepared. Structures were determined by 1D and 2D NMR and mass spectra. Compound 1 showed potent in vitro antifungal activity against Cryptococcus neoformans and Aspergillus fumigatus (IC(50) values = 0.4 and 3.0 microg/mL, respectively) and antibacterial activity against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare (IC(50) values of 0.35 and 0.9 microg/mL, respectively). The remarkable in vitro fungicidal activity of 1-4 against C. neoformans (MFCs = 0.63-1.25 microg/mL) and 2, 3, and 5 against A. fumigatus (MFCs = 0.63-2.5 microg/mL) were similar to amphotericin B, but >2-4-fold more potent than 6-8. Prosopilosidine (1) showed potent in vivo activity at 0.0625 mg/kg/day/ip for 5 days in a murine model of cryptococcosis by eliminating approximately 76% of C. neoformans infection from brain tissue compared to approximately 83% with amphotericin B at 1.5 mg/kg/day. Compounds 1 and 4 exhibited potent activity and high selectivity index (SI) values against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum, with IC(50) values of 39 and 95 ng/mL and 42 and 120 ng/mL, respectively (chloroquine, IC(50) = 17 and 140 ng/mL). Prosopilosine (1) also showed in vivo antimalarial activity, with an ED(50) value of approximately 2 mg/kg/day/ip against Plasmodium berghei-infected mice after 3 days of treatment.

Effect of green tea and its polyphenols on mouse liver.

Increased consumption of green tea (GT) without enough scientific data has raised safety concerns. Epigallocatechin 3-gallate (EGCG) is the most prominent polyphenol of GT that has antioxidant activity. However, higher doses of EGCG have been shown to cause liver injury. This study was initiated to determine the effect of GT extracts in a mouse model. We also investigated the effects of EGCG in normal and health-compromised mice. Different doses of GT fractions and EGCG were administered for 5 days to mice. Also, a single dose of lipopolysaccharide (LPS) was combined with EGCG in order to investigate its effect in the presence of fever. Plasma ALT and ALP levels were determined along with liver histopathology. Combining a single high IG dose of EGCG with a single IP dose of LPS initiated liver injury. Furthermore, repeated administration of high IG doses of EGCG showed mild liver injury, but it was augmented under febrile conditions induced by LPS. This study confirms the safety of reasonable consumption of GT over a short term. However, it highlights a caution that high doses of EGCG can lead to mild liver injury, and this may be markedly enhanced under febrile conditions.

Benzothieno[3,2-b]quinolinium and 3-(phenylthio)quinolinium compounds: Synthesis and evaluation against opportunistic fungal pathogens.

Substitution around 5-methyl benzothieno[3,2-b]quinolinium (2) ring system was explored in order to identify positions of substitution that could improve its antifungal profile. The 3-methoxy (10b) was active against C. albicans, C. neoformans, and A. fumigatus and the 4-chloro (10f) analog showed moderate increases in anti-cryptococcal and anti-aspergillus activities. The effectiveness of 10b and 10f were validated in murine models of candidiasis and cryptococcosis, respectively. The efficacy of 10f in reducing brain cryptococcal infection and its observation in the brain of mice injected with this quaternary compound confirm the capacity of these compounds to cross the blood-brain barrier of mice. Overall, several of the chloro and methoxy substituted compounds showed significant improvements in activity against A. fumigatus, the fungal pathogen prevalent in patients receiving organ transplant. Opening the benzothiophene ring of 2 to form 1-(5-cyclohexylpentyl)-3-(phenylthio)quinolinium compound (3) resulted in the identification of several novel compounds with over 50-fold increases in potency (cf. 2) while retaining low cytotoxicities. Thus, compound 3 constitutes a new scaffold for development of drugs against opportunistic infections.

Bioavailability, pharmacokinetics, and tissue distribution of the oxypregnane steroidal glycoside P57AS3 (P57) from Hoodia gordonii in mouse model.

P57AS3 (P57), an oxypregnane steroidal glycoside, is known to be responsible for the appetite suppressing activity of HOODIA GORDONII, a dietary supplement used for weight loss. In this study, bioavailability, pharmacokinetics, and tissue distribution of P57 were determined in CD1 female mice after administration of a single dose of enriched methanolic extract of HOODIA GORDONII (equivalent to a dose of 25 mg of P57/kg) by oral gavage or a single dose of purified P57 (25 mg/kg) intravenously. The level of P57 in plasma and tissues (brain, liver, kidney, and intestine) was determined by UPLC-MS. After oral administration of HOODIA extract, the peak plasma level of P57 was achieved in 0.6 h. Upon intravenous administration, the plasma clearance rate of P57 was 1.09 L/h/kg. P57 was rapidly distributed and eliminated from the tissues within 4 hours. The level of tissue distribution was highest in the kidney followed by liver and brain. Upon oral administration, P57 was not detected in the brain and a very low concentration was seen in the intestine, kidney, and liver. Tissue/plasma ratio was 0.33 for brain, 0.57 for liver, and 0.75 for kidney with IV route and 0.11 for intestine, 0.02 for liver, and 0.04 for kidney with oral route. The half-life of the elimination phase was similar with both routes. The oral bioavailability was 47.5 % and the half-life of the absorption phase was 0.13 h. In conclusion, P57 showed moderate bioavailability and was eliminated rapidly.

Tissue factor antisense deoxyoligonucleotide prevents monocrotaline/LPS hepatotoxicity in mice

Tissue factor (TF) is a membranous glycoprotein that functions as a receptor for coagulation factor VII/VIIa and activates the coagulation system when blood vessels or tissues are damaged. TF was upregulated in our monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity model. We tested the hypothesis that TF‐dependent fibrin deposition and lipid peroxidation in the form of oxidized low‐density‐lipoprotein (ox‐LDL) accumulation contribute to liver inflammation induced by MCT/LPS in mice. In the present study, we blocked TF using antisense oligodeoxynucleotides against mouse TF (TF‐ASO). TF‐ASO (5.6 mg kg−1) was given i.v. to ND4 male mice 30 min after administration of MCT (200 mg kg−1) p.o. followed after 3.5 h by LPS i.p. (6 mg kg−1). Blood alanine aminotransferase (ALT), TF, ox‐LDL, platelets, hematocrit and keratinocyte‐derived chemokine (KC) levels were evaluated in different treatment groups. Fibrin deposition and ox‐LDL accumulation were also analyzed in the liver sections using immunofluorescent staining. The results showed that TF‐ASO significantly restored blood ALT, hematocrit and KC levels, distorted after MCT/LPS co‐treatment, as well as preventing the accumulation of ox‐LDL and the deposition of fibrin in the liver tissues, and thereby inhibited liver injury caused by MCT/LPS. In a separate experiment, TF‐ASO administration significantly prolonged animal survival. The current study demonstrates that TF is associated with MCT/LPS‐induced liver injury. Administration of TF‐ASO successfully prevented this type of liver injury. Copyright

Tissue factor dependent liver injury causes release of retinoid receptors (RXR-α and RAR-α) as lipid droplets

Hepatic stellate cells (HSC) store retinoids and upon activation differentiate into myofibroblast-like cells, a process whereby they lose their retinoid-containing lipid droplets. We reported earlier, activation of tissue factor (TF) in our MCT/LPS hepatotoxicity model. We now report the involvement of TF in the release of retinoid receptors RAR-α and RXR-α as accumulated lipid droplet during monocrotaline/lipopolysaccharide (MCT/LPS)-liver injury. Constitutive expression of RAR-α was observed in HSCs and endothelial cells of bile duct and portal vein, while expression of RXR-α was observed in certain pericentral hepatocytes and HSCs. Administration of sub-toxic doses of MCT or LPS strongly increased TF and RXR-α but not RAR-α expressions in HSCs and hepatocytes. However MCT/LPS co-treatment showed insoluble droplets containing RAR-α and RXR-α in the vicinity of the necrotic areas. Blocking TF with TF antisense oligonucleotides (TF-AS ODN) led to normal hepatocyte expression of RXR-α and upregulated the expression of RAR-α in HSCs. This study shows clear evidence of in vivo release of RAR-α and RXR-α as insoluble lipid droplets in liver injury. It is possible that these insoluble droplets of RAR-α and RXR-α could be used as markers for liver injury in general and activation of HSCs in particular. RXR-α appears to be a more sensitive than RAR-α as it was affected by even the subtoxic doses of MCT or LPS. The fact that TF-AS treatment not only down-regulated TF but also obliterated the release of RAR-α and RXR-α as insoluble lipid droplets in hepatocytes points towards TF being an important regulatory molecule for RAR-α and RXR-α.

Stem cell intervention ameliorates epigallocatechin-3-gallate/ lipopolysaccharide-induced hepatotoxicity in mice

Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor β1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.

Thymoquinone Protects Against EGCG/LPS Induced Hepatotoxicity in Mice

Green tea (GT) is a widely used beverage and folk medicine. Epigallocatechin-3-gallate (EGCG), the main polyphenol of GT has been demonstrated to impact a number of biological targets. Our previous work has shown that the combination of LPS and EGCG in male mice causes hepatotoxicity, perhaps related to a prooxidant effect. Thymoquinone (TQ) is a prominent constituent of “black seed”, Nigella sativa, and is known for a number of biological activities, including potent antioxidant and anti-inflammatory activities. It has been extensively used in the Middle East as a protectant against hepatic and renal injuries. In the current study we treated mice with oral doses of TQ before and concurrently with EGCG/LPS. Thymoquinone ameliorated the rise in the plasma concentrations of ALT, ALP, TB, Amylase and the decrease of A/G ratio to normal after exposure to EGCG/LPS. It also blunted the weight loss. No histopathological lesions or mortality was observed. It can be concluded from this study that TQ showed a hepatoprotective effect in a murine model of EGCG/LPS induced hepatotoxicity. This hepatoprotective effect may be related to the antioxidant properties of TQ, but this conclusion needs further evaluation.

Efficacy of Prosopilosidine from Prosopis glandulosa var. glandulosa against Cryptococcus neoformans Infection in a Murine Model

In this study, 2,3-dihydro-1H-indolizinium alkaloid-prosopilosidine (PPD), that was isolated from Prosopis glandulosa, was evaluated against C. neoformans in a murine model of cryptococcosis. In vitro and in vivo toxicity of indolizidines were also evaluated. Mice were infected via the tail vein with live C. neoformans. Twenty-four hours post-infection, the mice were treated with PPD once a day (i.p.) or twice a day (bid) orally, or with amphotericin B (Amp B) intraperitoneally (IP), or with fluconazole (Flu) orally for 5 days. The brains of all of the animals were aseptically removed and the numbers of live C. neoformans were recovered. In vitro toxicity of indolizidine alkaloids was determined in HepG2 cells. PPD showed to be potent in vivo activity against C. neoformans at a dose of 0.0625 mg/kg by eliminating ~76% of the organisms compared to ~83% with Amp B (1.5 mg/kg). In addition, PPD was found to be equally efficacious, but less toxic, at either 0.125 or 0.0625 mg/kg compared to Amp B (1.5 mg/kg) when it was administered bid (twice a day) by an i.p. route. When tested by an oral route, PPD (10 mg/kg) showed potent activity in this murine model of cryptococcosis with ~82% of organisms eliminated from the brain tissue, whereas Flu (15 mg/kg) reduced ~90% of the infection. In vitro results suggest that quaternary indolizidines were less toxic as compared to those of tertiary bases. PPD (20 mg/kg) did not cause any alteration in the plasma chemistry profiles. These results indicated that PPD was active in eliminating cryptococcal infection by oral and i.p. routes at lower doses compared to Amp B. or Flu.

Quantification of mesembrine and mesembrenone in mouse plasma using UHPLC‐QToF‐MS: Application to a pharmacokinetic study

Sceletium tortuosum, is an indigenous herb of South Africa which is widely used as an herbal supplement in the treatment of anxiety and stress. Mesembrenone and mesembrine are the two main pharmacologically active alkaloids present in the extract. Despite the wide therapeutic applications of Sceletium extract, there are no reports of in vivo pharmacokinetic properties or analytical methods to quantify these two important alkaloids in plasma. Therefore, the current study aimed to develop and validate a simple and sensitive analytical method for simultaneous quantification of mesembrenone and mesembrine in mouse plasma. Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC/QToF-MS) was employed to achieve our objectives. The compounds were extracted using protein precipitation by methanol (100%) with quinine as an internal standard. The lower limit of quantification for both the compounds was 10 ng/mL. The extraction recovery was between 87 and 93% for both compounds with no matrix effects on the analysis. The accuracy was between 89.5 and 106% and precision was <12.6% for all quality control samples. This validated method was successfully applied to evaluate the i.v. plasma pharmacokinetics of mesembrine and mesembrenone in mouse. However, the oral bioavailability of these alkaloids was poor and the plasma levels were below the detection limits.