Landscape edging manufacturers EXTERNAL WORKS Landscape edging. Material; . Haddonstone's range of garden & landscape lawn edging . the company can produce bespoke landscape edging for creative design .
Garden Landscaping Materials For Sale - GardenSite.co.uk Shop by Material. Aluminium Garden Furniture . Border & Lawn Edging ; . our garden landscaping supplies can turn your ideas into reality.
66 Creative Garden Edging Ideas to Set Your Garden Apart 66 Creative Garden Edging Ideas. . So I've sourced 66 of the most creative garden edging ideas that will . it is the perfect edging material for traditional .
Amazon.co.uk: plastic garden edging Amazon.co.uk: plastic garden edging. . Plastic Flexible Lawn Edging . . MATERIAL: PLASTIC PVC NEW 4 PANELS GARDEN PICKET FENCING BORDER.
Garden Edging, Borders & Stepping Stones Wayfair.co.uk Shop Wayfair.co.uk for all the best Garden Edging. Enjoy Free Shipping on most stuff, even big stuff.
Using Landscape Edging - Better Homes and Gardens Landscape edging is a permanent, hard material that supplies a crisp edge between areas of the garden. Here's a guide to using landscaping edging in your yard.
Welcome - EverEdge - flexible metal garden edging and . EverEdge flexible steel lawn edging is the solution to the age old problem of maintaining neat edges for lawns, drives, paths, driveways and flower or vegetable beds.
Lawn Edging Materials: Pros, Cons of the Various Types What lawn edging materials are available to the average homeowner? Choices range from natural ones to manufactured materials; we discuss options here.
25 Garden Bed Borders, Edging Ideas for Vegetable and . 25 Garden Bed Borders, Edging Ideas for Vegetable and Flower Gardens. 25 Garden Bed Borders, Edging Ideas for . Brick landscape edging is the most popular option in .
Landscape Edging & Mow Strips - Landscaping Network Use this article, chart and photos to compare landscape edging options for your lawn or garden.
Lawn Edging Garden Borders - DIY Timber & sheet materials. Constructional timber; . This border edging is perfect for creating borders for flower beds or for path edging around the garden. .
Garden Edging & Borders - Wooden & stone edging at . Trying to be the envy of all your friends? Shop our wide range of lawn edging now, and seamlessly divide any garden space stylishly, by browsing Homebase.
The Best Garden Edging Tips — The Family Handyman These simple, attractive garden edging ideas will keep grass from invading your garden and eliminate the need for edge trimming.
37 Creative Lawn and Garden Edging Ideas with Images . 37 Creative Lawn and Garden Edging Ideas with . 37 Creative Lawn and Garden Edging Ideas with Images 1. Garden . can be excellent materials for your garden edging.
garden edging in Landscaping & Garden Materials eBay Find garden edging from a vast selection of Landscaping & Garden Materials. Get great deals on eBay!
Other Garden Edging - Plastic lawn edging & coping at . Spruce up your outdoor area this summer with our wide range of decorative plastic lawn edging and border roll. Buy in store and online now at Homebase.
Tips on Garden Border Materials DIY Edging materials improve the look of your garden beds, while keeping mulch and soil in and grass and gravel out. But what kind of edging is best?
Recycled Material Lawn Edging - Lawn Edging - Primrose Recycled Lawn Edging. Do your bit to save the environment with our plastic recycled lawn edging.


Salman Khan's production movie Notebook featuring Zaheer Iqbal and Pranutan Bahl was released this Friday. The box office collection of Notebook seems to have affected by Vidyut Jammwal starrer Junglee, which earned a decent amount at the ticket window. According to Box Office India report, Zaheer-Pranutan's Notebook started off on a poor note and managed to earn only 60-70 lakh on its opening day. "It is three times less than Junglee which itself is not that good," the report says.
Akshay Kumar's Kesari, which was released on 21 March, still ruling the box office and earned Rs 4.45 crore on March 29, which is better than Vidyut Jammwal's Junglee as well. The box office collection of Notebook is even lower than Salman Khan Production film Loveyatri, which released last year. The film Loveyatri, which featured Aayush Sharma and Warina Hussain earned Rs 2.11 crore nett on its day one.
Directed by ace filmmaker Nitin Kakkar, Notebook is basically based on the Thai film named A Teacher's Diary. The story of the film revolves around Kabir, an ex-army officer, who comes to teach students in Kashmir. He discovers a diary which was left behind by the previous teacher Firdaus, and later, he falls in love with her after learning her story. The movie has been getting positive reviews from all the quarters.
The film Notebook received positive reviews from both critics and audiences alike. In an interview with Mid-Day, Zaheer had revealed how he bagged the role. "Notebook happened to me while I was dancing at my sister's wedding at her sangeet performance. We had shot a video of a story where my sister and brother-in-law win the local best couple award in 2025, and I was playing my brother-in-law's part and every time the song appeared in the video, I had to come on stage and dance to it. When I got off the stage, Salman (Khan) bhai happened to be there because he is my dad's childhood friend. He wasn't supposed to be there since he had a court case in Jaipur but that got pushed," he narrated.


text:
Derek Bell may refer to:

Sports

 Derek Bell (baseball) (born 1968), American baseball player
 Derek Bell (footballer, born 1956), English former professional footballer
 Derek Bell (footballer, born 1963), English former professional footballer
 Derek Bell (racing driver) (born 1941), British racing car driver

Other
 Derek Bell (musician) (1935–2002), Northern Irish musician and composer
 Derek Bell (physician), professor of acute medicine

See also
 Derrick Bell (1930–2011), American lawyer, professor, and civil rights activist

redpajama_set_name: RedPajamaWikipedia


text:
Special Protection Areas and Districts-
Neighborhood Conservation District (NCD) Zoning Overlays-
CD-8: Glen Lennox Area +
Glen Lennox NCD: Background and Process
Town Hall » Departments & Services » Planning » Special Protection Areas and Districts » Neighborhood Conservation District (NCD) Zoning Overlays
Pine Knolls Neighborhood Conservation District (CD-4)
The Pine Knolls Neighborhood has been designated by the Town Council as a Neighborhood Conservation District zoning overlay.
Notice: Some elements of the Pine Knolls Neighborhood Conservation District are no longer enforceable by the Town due to the enactment of North Carolina Session Law 2015-86, which became law on June 19, 2015.
For more information about the specific elements affected, click here.
Click here for a copy of the bill.
For a link to an article from the UNC School of Government about Senate Bill 25 (posted August 3, 2015), click here.
For a video of the August 26, 2015 Public Information Meeting, click here.
For questions, contact Jay Heikes, Planner, at 919-969-5082 or jheikes@townofchapelhill.org
The Pine Knolls Neighborhood was the fourth neighborhood in Chapel Hill to become a Neighborhood Conservation District; therefore, it is referenced as CD-4.
The following is a map of the Pine Knolls Neighborhood Conservation District zoning overlay:
What is a Neighborhood Conservation District Zoning Overlay?
Neighborhood Conservation District (NCD) Zoning Overlay is a set of land use regulations that is applied to a specific neighborhood as a zoning overlay. Any new construction or additions must comply with the Neighborhood Conservation District regulations.
For more information about NCD zoning overlays, click here.
Pine Knolls Neighborhood Conservation District and Design Guidelines
On June 12, 2006, the Town Council amended the Zoning Atlas to include the Pine Knolls Neighborhood Conservation District Zoning Overlay. On January 9, 2012, and again on June 25, 2012, the Pine Knolls Neighborhood Conservation District regulations were amended in conjunction with the creation of the Northside and Pine Knolls Community Plan.
For an overview of the NCD regulations, click here: Pine Knolls Neighborhood Conservation District Regulations
For an overview of the recommended design guidelines, click here: Pine Knolls Design Guidelines
Are you planning any new construction or additions to a property within the Pine Knolls Neighborhood Conservation District?
Any new construction or additions must comply with the Pine Knolls Neighborhood Conservation District regulations.
If you have questions, please contact the Town's Development Services Division at 919-969-5066. The Development Services Desk is located on the 1st floor of Town Hall, 405 Martin Luther King Jr. Blvd., Chapel Hill, 27514.
Permits Needed for Construction
For any type of construction (including additions and renovations) to your house or property, it is most likely necessary to obtain a permit from the Development Services Division of the Office of Planning and Sustainability. Prior to beginning any work on your property, please contact the Development Services Division.
For the Staff Evaluation Form, click here: Regulations and Standards Application for Single-Family
In general:
Projects that add less than 250 square feet of floor area require a Single-family Zoning Compliance Permit (some projects may only need a Single-family Express Zoning Compliance Permit).
Projects adding more than 250 square feet of floor area or proposing demolition need approval from the Town's Planning Board and should use the form for NCD Site Plan Review.
Projects adding more than 600 square feet of floor area must first present a Concept Plan to the Town's Community Design Commission using the form for NCD Concept Plan Review and must also obtain approval of a Site Plan from the Town's Planning Board as described above.
Submittal Deadlines for Commissions
For Pine Knolls submittal deadlines for the Planning Commission, click here.
For Pine Knolls submittal deadlines for the Community Design Commission, click here.
For more information about the Northside and Pine Knolls Community Plan, click here.
For a copy of the staff memorandums to Town Council:
Memorandum titled "Proposed Rezoning to Create a Neighborhood Conservation District for the Pine Knolls Neighborhood" - June 12, 2006 - click here.
Memorandum titled "Consideration of Northside and Pine Knolls Community Plan and Proposed Amendments to the Northside and Pine Knolls Neighborhood Conservation Districts" - January 9, 2012 - click here.
Memorandum titled "Consider Amendments to the Land Use Management Ordinance, the Northside and Pine Knolls Neighborhood Conservation Districts and the Comprehensive Plan" - June 25, 2012 - click here.
For more information about other Special Protection Areas in Chapel Hill, click here.

redpajama_set_name: RedPajamaCommonCrawl


MacGurus Technical Forums > General Hardware and Software Discussions > Video Upgrades > ATI Radeon driving 2 displays = i controller or 2 w/pci bridge?
View Full Version : ATI Radeon driving 2 displays = i controller or 2 w/pci bridge?
Does the ATI Radeon series of PCI card have one controller capable of driving two displays, or are there two controllers with a PCI Bridge?
I want to drive a pair of Apple Studio Display LCDs from a single PCI card in a 20th Anniversary Mac (TAM). Like the 6500, the TAM has a buggy PCI controller, which is unfriendly to PCI bridges as found in USB+FW cards and expansion chassis.
How do you tell which is which? I have a Radeon 7K coming in in a few days and can check for you.
There may not be any way to tell from examination of the card. Apple System Profiler or PCI Prober might provide some insite though.
While some bridge bearing cards are made with a separate bridge chip and then two of whatever device it supports (or separate Firewire and USB chips for the combo cards) it is possible that ATI integrated a Bridge and two Radeon chips all on the same piece of silicon.
The ATI RAGE128 VR and the old RADEON support dual displays - via the S-Video. I think the RADEON 7K is the same - more or less, but it has two VGAs out and adaptor(s) for hooking it to DVI, VGA or sandard S-Video I think.
The 3Dfx V5 has a bridge chip in it - pci bridge to be more accurate. It only supports one monitor though.
Is that why I can run two monitors with the Voodoo5: because I'm using the mobo VRAM in my 8500 to run the second one?
Thanks for all the responses, especially regarding the Voodoo bridge.
I'll spend a few more bucks to get a Radeon 7000 locally so that i can take it back without a hassle if it doesn't work..
My VooDoo 5 card used to show up as 2 cards in Apple System Profiler. I had it in a B&W, they had 4 PCI slots and ASP would show 5 PCI slots is this because of the bridge?
Anyway the Radeon 7000 only shows as one card in ASP.
I wonder if this is due to the dual processors?
yeh mine does almost the same...except i have it in slot a1 and in the second a1 info box my card model is a "-1" and the card revision is "not available"... i thought it was odd when i first got this card but didn't know enough to say it was odd but now that others have come out i think i'll say it is odd...odd huh?
So I installed a Radeon 7000 in my Twentieth Anniversary Macintosh (TAM) with limited success. In 1 hour of testing things appear OK if you want VGA, SVIDEO or both simultaneously, using OS9.1 on legacy PCI Mac.
TAM configuration before Radeon 7000 install. 128M RAM, Toshiba 30G HD, Sonic CSII Ethernet, with Sonnet Crescendo L2 G3-500, Rage II motherboard video driving a built-in 800x600 LCD. ATI drivers were present from MacOS install, plus I was surprised to find that the 9.1 install CD from a 533-dual G4 Digital Audio had already installed ATI's "minimum" OpenGL v1.2.2 with 9.1. However I upgraded to OpenGL v1.2.4 copied from a Pismo 9.2.2 extensions folder.
I was Mighty Pleased(tm) to discover the Radeon 7000 does not have a fan, but rather a low-profile heat sink!. My TAM is nearly silent, and does not accommodate a very thick PCI card. I was not looking forward to additional noise in my environment, nor leaving the case open. At least one support chip becames too hot to touch, which was a painful discovery. I may add a heat sink to that chip to aid system longevity.
The Radeon 7000 docs state clearly and often that the user should install the ATI software before the PCI card. I have contempt for hardware that crashes without drivers, so I installed the 7000 card with two VGA LCDs, using both the VGA (Display #2) and DVI (Display #3) connectors of the 7000, since ATI kindly provided a DVI-to-VGA adapter amongst others.
I rebooted, resetting PRAM from keyboard three times before allowing full startup.
The TAM crashed on boot, at the 9.1 ATI Graphics Extension, reporting a System Error "Illegal Instruction"/Restart dialog. When I clicked OK, the machine repeated the same warning, but changed from Illegal Instruction to "Error Type 10" [sdmJTInitError - SDM Jump Table could not be initialized] and froze utterly, requiring a 3 finger restart.
Rebooting with the shift key was successful. The TAM internal video was fine, and the LCD 2 on the 7000's VGA port showed the extended desktop. Oddly, the LCD 3 on the DVI port showed a green (good sync) LED, but remained black, displayed no image. Monitors control panel was unlaunchable. However, a video utility i wrote back in the NuBus days, ftp://soundmark.net/pub/mac/WindowMaker4.bin , beeped 3 times, indicating I had 3 video devices recognized!
I disabled all ATI extensions and rebooted to 9.1 successfully. The monitors control panel confirmed that I had 3 screen devices, and the green LED was lit, but DVI still displayed no image. I installed the Quicktime 5.0.2 and ATI drivers from the 7000 CDROM and rebooted.
There was still no display on the DVI-connected LCD #3, still the green sync OK LED was lit. VGA display worked as expected, and SVIDEO output worked as expected. However, even with ATI drivers installed, DVI+VGA and DVI+SVIDEO and DVI alone did not produce image on the DVI-VGA screen. I had to go to work before I could test 3D gaming perfomance.
So far I have no joy running two monitors from the card like I hoped. Preliminary results indicate that I can run VGA and SVIDEO simultaneously, but SVIDEO is not why i spent my $116.
- Testing the 7000 with ATI cards Rage 3D PRo and Mach64 in multiple PCI slot Macs.
I haven't tried that yet w/ my radeon - at the moment, I have it connected up to a SGI monitor and the Rage 128 that came w/ my Yikes is connected to a 19" Hitachi.. Maybe I'll try doubling it out..
Damn, damn, damn! ATI says that legacy Apple monitor sense is NOT supported on Mac Radeon DVI-VGA adapters. ATI has no intention to support my two Apple VGA Studio Display LCDs on a Radeon 7000.
Door #1 - refund for Radeon 7000.
anyone have experience with MacWarehouse and RMAs?
Door #3 - Trade Apple VGA LCD for DVI LCD or non-Apple LCD.
a third PCI video card in an environment with only two displays.. great.
You may have bigger problems than a Mac Video Sense code issue.
Isn't the TAM based on the 6400 or 6500 design? It is my understanding that the 6400/6500 does not work properly with PCI-PCI Bridges. And, being a dual function card, there is a high probability that the R7000 has a PCI-PCI Bridge built in somewhere. So you could be experiencing the 6400/6500 incompatibility with PCI bridges.
I'm not certain about that. I have never personally tested a 6400/6500, and I don't *know* that the R700 has a PPB built in, but that is my hearsay based understanding.
If the tone of this post seems peeved, it's because I'm annoyed at ATI and MacWarehouse. My good humor is strained by hours of unsuccessful testing, and i'm too stupid to go to bed without venting. Advance apologies, Trag; I'm not aiming at you.
Yes, the TAM shares the same motherboard with a 6500 or 6400, I forget. However, the TAM successfully drives two displays as long as neither is DVI, as decribed earlier. If there were two controllers behind a PCI bridge, the TAM would drive neither display. To wit, a generic USB+FW dual function card in the TAM failed to enable either USB or FW functionality despite successful add-in card driver installation.
Another consideration is that ATI's OEM 7500 AGP card uses the same controller of the retail 7000 PCI. ATI cannot afford to cripple AGP performance--even on a low-end controller--with an internal PCI bridge between it and the AGP bus--no way. NVIDIA's TwinView architecture doesn't use a PCI bridge; ATI's dual-monitor technology would not be able to compete with--or prevail in testing--against NVIDIA's AGP cards if ATI AGP bandwidth was limited by a PCI bridge.
Finally, my issue is virtually proven to be a monitor-sense/DDC issue. I came here this morning in the wee hours to report the Radeon 7000 didn't drive the DVI-VGA adapter properly in my 9500, my other 9500, or my Beige G3, none of which have the PCI bridge issue of the TAM/6x00.
I thought computers were supposed to make my life easier. Grumble grumble.
No offense taken, Ricercar, and, in fact, I appreciate the info that there isn't a PPB in the R7000. I've been wonderign about that but unable to confirm it one way or the other from any source.
That gives hope that the thing might work in the lower slots of the S900/J700, though it still may not.
Does the UMax S900 have the G4 PCI slot issue that the 9500/9600 has?
No, the S900/J700 does not have the G4/lower three slot issue. That issue appears to only occur in machines with dual Bandits.
However, the S900/J700 have a plethora of lower slot problems (any but the top two slots) related to those slots being behind a PPB.
My tests imply to me that the PowerSurge architecture (Powersurge = x500, x600, PTP, PW, S900/J700) has a PCI implementation bug which does not handle more than one level of PPB properly. So the lower slots of the S900/J700 mostly work correctly, but if you install a card with a PPB on board creating a hierarchy of two PPBs the machine will freeze during PCI initialization in most cases. There are some exceptions that aren't worth going into unless you're really interested. This also implies that the PowerSurge architecture is not actually PCI compliant as Apple claimed...unless the PCI-Bridge supplement came later than the PCI 2.0 standard from which Apple appears to have worked.
It sounds like you have (had?) some card designing experience or at least software experience. I'd be happy to read any thoughts you have on the topic.
hello trag... what is a bandit? Is it an MPC106 controller aka Grackle from Moto?
The Bandit is commonly called the PCI controller in the PowerSurge machines. These machines predate the Beige G3 by one generation. I believe that the Grackle (MPC 106) integrates both a memory controller and PCI controller onto one chip. But at any rate, they're definitely not the same. The Bandit was a custom chip design for/by Apple.
In the PowerSurge architecture, the top level bus, the CPU bus, has the CPU(s), the Hammerhead Memory controller, the Bandit(s) and in the 75/76/73/85/8600 machines, a CHAOS video controller.
The Hammerhead arbitrates who gets control of the address adn data busses (memory, CPU, Bandit 1, Bandit 2 or CHAOS) at any given moment. The CPU is the CPU and of course there may be one, two or four of them.
The Bandit is called a PCI controller but it is really more properly called a Bridge. The Bandit bridges data from the CPU bus to the PCI bus. So on one side the Bandit shares a bus with the CPU and memory and operates at your CPU bus speed. On the other side, the Bandit shares a bus with your PCI cards and operates at 33 MHz.
The CHAOS is analogous to the Bandit, but it's downstream side has built-in video circuitry instead of PCI devices.
The Beige G3 has a similar architecture, except the Hammerhead and Bandit is combined into the Grackle chip. There is no Chaos, because the built-in video is just another PCI device on the PCI bus. It's just soldered down, instead of in a PCI slot.
According to Apple's documentation, their design allowed for up to four Bridges on the CPU bus. So, in theory, there could be four Bandits (at least 12 PCI slots) or three Bandits and a CHAOS. It is also true that a Bandit can support more than three PCI slots. On the Apple Network Server, Bandit 2 supports four PCI slots, and Bandit 1 supports six PCI devices, though only two of them are actual slots.
So, in theory, one could build a PowerSurge machine with four Bandits, each supporting six PCI slots (well one of them would need to only have five because of the Grand Central Chip). So that would be 23 PCI slots.
In reality, it's unclear whether the Hammerhead has arbitration lines for four Bandits (does the CHAOS in a 7500 use the same arbitration lines as the second Bandit in a 9500--I don't think so, but am unsure). It's unclear whether there are interrupt lines available on Grand Central (interrupts go through Grand Central) to support additional PCI slots. I don't know enough about Open Firmware to tell whether you'd have to tell the machine that the extra slots exist and which interrupt lines they're connected to. There are Aliases built into the ROM of the PowerSurge machines but they only cover the two Bandits and the normal six PCI slots. It's unclear whether the ROM of the PowerSurge would be able to connect drivers for PCI cards up to the memory space for PCI slots on additional Bandits.
But if all the "ifs" above could be satisfied, one could build a machine with a lot of slots. It'd have slow memory by today's standards, but what a lot of slots.
With thanks. If the Grackle aka MPC106 is a bridge and memory contrller how does this relate to the jumper block at J16 in the Beige G3s (and the B&Ws). Some confusion abounds. Some appear to have suggested that the jumper 5-6 sets the "grackle" (ie Bekkome's site). If true the question becomes "Sets it to do what?". I don't get it... And then there is all sorts of stuff at the Moto site regardeing PLL, VCO and PCI. Again... I don't get it.
All I know is that my voodoo 5500 does funny stuff in my G3 when ASP looks at it.
I don't know much specific stuff about the Beige and the Grackle, but from what little I read I believe those jumpers set various clock speed ratios and the voltage level supplied to the CPU.
For example, PCI runs at 33 MHz. So there may be a 33 MHz clock on the Beige's motherboard. With one jumper setting you might run the CPU/memory bus speed at 2X the PCI speed or 66 MHz. With another setting you get 2.5X or 83 MHz. Similarly for the ratio between CPU speed and bus speed. Various jumper settings might provide a 5X CPU to bus ratio, or 5 X 66 = 330 MHz. Another setting might get you 6X or some such. I'm not actually sure whether the jumper block on the Beige has a CPU ratio setting or whether that's on the ZIF. But I am sure the Grackle has a PCI:CPU Bus speed ratio setting which is on that jumper block.
FWIW I'm totally unsurprised each time Apple has PCI bridge issues. They've screwed up _something_ in the PCI implementation nearly every generation since VillageTronics discovered the first-generation PCI macs were incompatible--in certain slots--with my then-employer's bi-endian video controllers.
7-9 sets the PCI clock spd?
5-6 sets the PCI to system bus ratio?
1-4 sets the PLL? or is this wrong? 1-4 appears to set the multiple/ratio used to determine the cpu clock based upon the selected ratio and the system bus speed.
So I'm buying Gregory's 15" Apple DVI LCD. Looks like I"l be able to put that second external display on my 20th Anniversary Mac after all.
Worked really well - except that I'm playing with HUGE graphics files (i.e molecular models etc) and having the VRAM halved really screwed up the ability to roll things around quickly.. so I reverted back and now have the Hitachi driven by the Radeon and the SGI under the stock card.
Chris, if you're just getting some wiggling like near one of the edges of the display, that may be a capacitor that needs replacing. That's fairly easy to do, but the trick, of course, is knowing which one to replace. If you're so inclined, you might take a look around http://www.repairfaq.com. There's a bunch of stuff there on repairing monitors and other things.
Oh, and don't electrocute yourself. :-) Insert standard warning about high voltages present inside monitors and TV sets.
Thanks Trag. A colleague basically gave me this display - it's odd - I can make it "not shake" if I drive it at a higher scan rate...I think it was actually meant to be an RMA to SGI but they (SGI) didn't seem to care about getting it back, so I ended up with it.
I hooked up a DVI and a VGA to my 20th Anniversary Macintosh, and they both operate as expected: Three displays, one 20th anniversary Mac.
*sigh.* Dreams do come true.
Apple System Profiler reports video memory as the expected 32M RAM, and adds "LCD and CRT displays in use." which I have never seen before.
On the devices and Volumes page ASP calls the Radeon 7000 a display card, but on the description it's ATY,DDParent and ATY, RC100Parent, and shows only one display connected. I wonder how the child is connected. The display reported is the DVI, not the VGA.
When I switched the VGA resolution from 1024x768@75Hz to 832x624@75Hz, the Studio Display began to go black for 0.5 seconds in 2 second intervals. I'll follow up here if I find an explanation or cause.


UFP Technologies offers complete turnkey solutions for packaging projects. From packaging design to drop-shipping we will work with you from start to finish.
We provide a number of medical device clients with design and engineering support, fabrication, packaging with their labels and drop shipping. We also provide kitting services for a number of protective case and insert customers. After manufacturing and assembly a customer's protective cases and inserts we can place their products in the cases before shipping.


text:
Research ArticleSpecial Section on Transporters in Drug Disposition and Pharmacokinetic Prediction
A Clinical Quantitative Evaluation of Hepatobiliary Transport of [11C]Dehydropravastatin in Humans Using Positron Emission Tomography
Ken-ichi Kaneko, Masaaki Tanaka, Akira Ishii, Yumiko Katayama, Takayoshi Nakaoka, Satsuki Irie, Hideki Kawahata, Takashi Yamanaga, Yasuhiro Wada, Takeshi Miyake, Kota Toshimoto, Kazuya Maeda, Yilong Cui, Masaru Enomoto, Etsushi Kawamura, Norifumi Kawada, Joji Kawabe, Susumu Shiomi, Hiroyuki Kusuhara, Yuichi Sugiyama and Yasuyoshi Watanabe
Drug Metabolism and Disposition May 2018, 46 (5) 719-728; DOI: https://doi.org/10.1124/dmd.118.080408
Ken-ichi Kaneko
RIKEN Center for Life Science Technologies (K.K., Y.K., T.N., S.I., Y.Wad., Y.C., Y.Wat.) and RIKEN Center for Molecular Imaging Sciences (Y.K., Y.Wad., Y.C., Y.Wat.), Chuo-ku, Kobe, Hyogo, Japan; Departments of Physiology, Nuclear Medicine, and Hepatology, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka, Japan (M.T., A.I., H.K., T.Y., M.E., E.K., N.K., J.K., S.S.); Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Bunkyo-ku, Tokyo, Japan (T.M., K.M., H.K.); and Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Tsurumi-ku, Yokohama, Kanagawa, Japan (K.T., Y.S.)
Masaaki Tanaka
Akira Ishii
Yumiko Katayama
Takayoshi Nakaoka
Satsuki Irie
Hideki Kawahata
Takashi Yamanaga
Yasuhiro Wada
Takeshi Miyake
Kota Toshimoto
Kazuya Maeda
Yilong Cui
Masaru Enomoto
Etsushi Kawamura
Norifumi Kawada
Joji Kawabe
Susumu Shiomi
Yasuyoshi Watanabe
Various positron emission tomography (PET) probes have been developed to assess in vivo activities in humans of drug transporters, which aid in the prediction of pharmacokinetic properties of drugs and the impact of drug-drug interactions. We developed a new PET probe, sodium (3R, 5R)-3, 5-dihydroxy-7-((1S, 2S, 6S, 8S)-6-hydroxy-2-methyl-8- ((1-[11C]-(E)-2-methyl-but-2-enoyl) oxy) -1, 2, 6, 7, 8, 8a-hexahydronaphthalen-1-yl) heptanoate ([11C]DPV), and demonstrated its usefulness for the quantitative investigation of Oatps (gene symbol SLCO) and Mrp2 (gene symbol ABCC2) in rats. To further analyze the species differences and verify the pharmacokinetic parameters in humans, serial PET scanning of the abdominal region with [11C]DPV was performed in six healthy volunteers with and without an OATP1Bs and MRP2 inhibitor, rifampicin (600 mg, oral), in a crossover fashion. After intravenous injection, [11C]DPV rapidly distributed to the liver and kidney followed by secretion into the bile and urine. Rifampicin significantly reduced the liver distribution of [11C]DPV 3-fold, resulting in a 7.5-fold reduced amount of excretion into the bile and the delayed elimination of [11C]DPV from the blood circulation. The hepatic uptake clearance (CLuptake, liver) and canalicular efflux clearance (CLint, bile) of [11C]DPV (544 ± 204 and 10.2 ± 3.5 µl/min per gram liver, respectively) in humans were lower than the previously reported corresponding parameters in rats (1800 and 298 µl/min per gram liver, respectively) (Shingaki et al., 2013). Furthermore, rifampicin treatment significantly reduced CLuptake, liver and CLint, bile by 58% and 44%, respectively. These results suggest that PET imaging with [11C]DPV is an effective tool for quantitatively characterizing the OATP1Bs and MRP2 functions in the human hepatobiliary transport system.
Multispecific transporters, such as organic anion–transporting polypeptide (OATP) 1Bs (gene symbol SLCO) and multidrug resistance–associated protein 2 [MRP2 (gene symbol ABCC2)], participate in hepatic uptake and canalicular efflux, respectively, to facilitate the excretion of a wide variety of organic anions into the bile from the blood circulation in humans (Shitara et al., 2005; Nies and Keppler, 2007; Maeda and Sugiyama, 2008; Yoshida et al., 2013). The direct measurement of drug concentrations in organs/tissue in vivo is a prerequisite for the functional characterization of drug transporters and the accurate prediction of the intrinsic clearance for overall elimination. Pharmacokinetic analyses based on plasma concentration profiles of drugs have been widely used to evaluate the overall intrinsic hepatic clearance mediated by these drug transporters. However, the elements of the overall intrinsic hepatic clearance, such as the sinusoidal uptake and the sinusoidal and canalicular efflux, are difficult to evaluate without knowing the tissue concentration time profiles of the drug in vivo. Furthermore, pharmacokinetic analyses involving the tissue concentration of a drug help to improve the quantitative investigation of drug-drug interactions (DDIs). The plasma concentrations of some drugs, the hepatic uptake of which is the rate-determining process, are not appropriate indices for assessing the changes in canalicular efflux transporters. Under such conditions, changes in the canalicular efflux transporters hardly affects the intrinsic clearance for the overall hepatic elimination, whereas liver concentrations are more profoundly altered than the plasma concentrations, depending on the changes (Watanabe et al., 2010; Maeda, 2015).
Positron emission tomography (PET) is a powerful method for quantitatively investigating the in vivo tissue distribution of drug candidates at a microdose level because of its high sensitivity and spatiotemporal resolution (Willmann et al., 2008; Kusuhara, 2013), thereby enabling the safe assessment of the pharmacokinetics of drug candidates in humans. We have established several PET probes for assessing drug transporters and demonstrated their usefulness in pharmacokinetic and DDI studies in animals (Takashima et al., 2010, 2011, 2013; Shingaki et al., 2013) and humans (Takashima et al., 2012). However, the functional characterization of multispecific drug transporters and the DDIs in vivo have not been fully analyzed to ensure the accuracy of pharmacokinetic prediction. For example, the (15R)-16-m-[11C]tolyl-17, 18, 19, 20-tetranorisocarbacyclin methyl ester (15R-[11C]TIC-Me), which was expected to be a PET probe for hepatobiliary transport, was metabolized into at least four metabolites within 20 minutes after administration (Takashima et al., 2012). Since the complicated metabolism of 15R-[11C]TIC-Me would decrease the evaluation accuracy of drug transporter functions, nonmetabolizable PET probes are urgently required.
We recently developed a new PET probe, a pravastatin derivative, dehydropravastatin [(3R, 5R)-3, 5-dihydroxy-7-((1S, 2S, 6S, 8S)-6-hydroxy-2-methyl-8-((1-[11C]-(E)-2-methylbut-2-enoyl) oxy)-1, 2, 6, 7, 8, 8α-hexahydronaphthalen-1-yl) heptanoate ([11C]DPV)] (Fig. 1), for the quantitative characterization of the Oatps and Mrp2 functions in the hepatobiliary transport (Ijuin et al., 2012). The reliability of [11C]DPV for the quantitative estimation of the Oatps and Mrp2 functions has already been demonstrated in rats using rifampicin or in Mrp2-deficient mutant rats (Shingaki et al., 2013). Rat hepatocytes produced a metabolite from [11C]DPV, and human hepatocytes produced no metabolites (Shingaki et al., 2013). This finding suggests that [11C]DPV is a useful PET probe for the precise evaluation of hepatobiliary transport in animals and humans.
Chemical structure of [11C]DPV.
We performed a clinical PET study with [11C]DPV as a nonmetabolizable PET probe in healthy volunteers to assess the feasibility of using [11C]DPV in the pharmacokinetic analysis in humans. The utility of [11C]DPV PET imaging for the quantitative investigation of the drug transporters in humans was also examined using rifampicin, a well-known drug causing OATP1Bs-mediated DDIs. In addition, to confirm the membrane transport by OATP1Bs in the liver, an in vitro uptake study of DPV using OATP1B1- and OATP1B3-expressing cell lines was also conducted.
[11C]DPV (Fig. 1) was synthesized at Osaka City University Hospital in accordance with the procedure reported previously (Ijuin et al., 2012). The purity and concentration of [11C]DPV were assessed by high-performance liquid chromatography. The synthetic procedure for [11C]DPV was validated at Osaka City University. The radiochemical purity was 100% (n = 3), and the specific radioactivity was 5.4–35.6 GBq/µmol at the time of injection. The purified fraction was evaporated and reconstituted with 5 ml of saline.
[3H]Estradiol-17β-glucuronide (E217βG; 40 Ci/mmol, >97%) was purchased from PerkinElmer (Waltham, MA). Rifampicin was purchased from Sigma-Aldrich (St. Louis, MO).
Subjects and Study Design.
The study was a single-center (Osaka City University Hospital), single-dose (microdose) clinical study. The study protocol was approved by the Ethics Committee at Osaka City University. All subjects provided their written informed consent before the study. Six healthy male Japanese subjects and a patient with Dubin-Johnson syndrome (DJS) diagnosed based on the clinical features and serum bilirubin species (Dubin and Johnson, 1954; Lee et al., 2006; van Dijk et al., 2015) were enrolled in the study. The mean ages in the healthy subjects and the DJS patient were 32 ± 9 and 67 years; the body weight and height in the healthy subjects were 65.3 ± 8.7 kg and 166.8 ± 6.1 cm, respectively, and that in the DJS patient were 59 kg and 158 cm, respectively. The subjects were allowed water but refrained from food intake for half a day before the study. Each healthy subject was enrolled for two study periods, and the DJS patient was enrolled for the first study period. In the first study period, a single intravenous bolus of [11C]DPV was administered. In the second study period, a single intravenous bolus of [11C]DPV was administered 1 hour after the oral administration of rifampicin (Rifadin; Daiichi-Sankyo, Tokyo, Japan) at a therapeutic dose of 600 mg (four 150-mg capsules). The dose of [11C]DPV administered per body was 85.5 ± 12 MBq (chemical amount per body, 6.8 ± 4.3 nmol).
PET Scans.
Dynamic scanning was performed with a PET/computed tomography Biograph Scanner (Siemens, Knoxville, TN). Before PET scanning, each subject was placed in a supine position on the scanner bed with belts attached to minimize the body movement during scanning, and then the right and left median cubital veins were cannulated for radiotracer injection and blood sampling, respectively. At the start of PET scanning, [11C]DPV was intravenously administered for 30–60 seconds, and then the catheter line was flushed with 10–25 ml of saline to prevent radiotracer retention in the cannula. Serial PET scanning of the abdominal region was performed over 90 minutes in the three-dimensional dynamic mode in the following sequence: 12 × 5, 8 × 15, 4 × 30, 5 × 60, 5 × 120, 2 × 300, and 3 × 600 seconds. Blood was drawn a maximum of 13 times (at 0, 1, 2, 3, 4, 5, 6, 8, 10, 20, 30, 45, and 60 minutes after radiotracer administration). The volume of blood sampled at 0 minutes was approximately 20 ml and that at the other time points was approximately 1.5 ml. The amount of radioactivity in each sample was determined with a well-type γ-counting system (well-stand type FS-3-A; Shimadzu, Kyoto, Japan), and the data were corrected for any time decay from the point of radiotracer administration.
The Uptake of DPV by OAPT1B1- and OATP1B3-Expressing HEK293 Cells.
An in vitro uptake study of DPV using empty vector–transfected and OATP1B1-expressing cells was conducted at Tsukuba Research Institute (Sekisui Medical Co. Ltd., Tokyo, Japan). The cells had been established as stably expressing OATP1B1 after lipofection of pcDNA3.1 containing an open reading frame of OATP1B1 (NM006446). An in vitro uptake study of DPV using empty vector–transfected, and OATP1B3-expressing cells was conducted as previously reported (Hirano et al., 2004, 2006; Matsushima et al., 2005; Kitamura et al., 2008). The amount of DPV in the uptake buffer and the cell specimens was quantified using an AB SCIEX QTRAP 5500 Mass Spectrometer (Applied Biosystems, Foster City, CA) equipped with a Prominence high-pressure liquid chromatography system (Shimadzu) operated in the negative electrospray ionization mode as previously reported (Shingaki et al., 2013). The transporter-specific uptake was determined by subtracting the uptake into vector-transfected cells from the uptake into complementary DNA-transfected cells.
Analysis of PET Imaging Data.
Acquired PET image data were normalized, and all appropriate corrections were made for dead time, attenuation, and scanner calibration. The emission images were reconstructed by Fourier rebinning and two-dimensional filtered back projection with a ramp filter that had a cutoff at the Nyquist frequency (0.0 mm). Regions of interest were delineated for the liver, kidneys, bile duct, and gallbladder, which were visually identifiable, using PMOD 3.0 software (PMOD Technologies LLC, Zürich, Switzerland). For the liver and kidney images, the regions of interest were defined by calculating the radioactivity concentration and the total amount of radioactivity in the tissues. For the gallbladder and bile duct images, regions of interest that were slightly larger than the images but not overlapping the images of the other tissues, such as the liver and kidneys, were defined to calculate the total amount of radioactivity in the tissues. In this analysis, the radioactivity in the gallbladder and bile duct corresponded to that excreted into the bile. Time-radioactivity curves for each type of tissue were constructed by normalizing the decay-corrected time-radioactivity measurements to the injected dose of [11C]DPV.
Radiometabolite Analyses.
Radiometabolite analyses of human blood were performed using the sample obtained 2, 5, 10, 20, and 30 minutes after radiotracer administration for the PET study to confirm the production of the radiometabolites. The sample was deproteinized by precipitation with an equivalent volume of acetonitrile and centrifuged at 12,000 rpm for 2 minutes at room temperature. The supernatants of blood samples were applied to RP-8 thin layer chromatography (TLC) plates (Merck Millipore, Darmstadt, Germany). The plates were developed at room temperature with acetonitrile/water/formic acid (50:50:1) as a mobile phase. After migration, the plates were dried and exposed to BAS TR2040 imaging plates (Fujifilm Corporation, Tokyo, Japan) for 40 minutes. The distribution of radioactivity on the imaging plates was determined by digital photostimulated luminescence autoradiography using a Fuji FLA-7000 analyzer (Fujifilm Corporation) at 50-μm resolution. The data were then analyzed using MultiGauge image analysis software (Fujifilm Corporation) to calculate the proportion of parent [11C]DPV.
In Vivo Kinetic Analyses to Determine Clearance on PET Imaging.
The initial [11C]DPV uptake clearance was calculated for the human liver or kidneys by the integration plot method (Kim et al., 1988), using the portion of time-radioactivity curves encompassing the linear range of the plot after [11C]DPV administration, during which the effects of the metabolism and excretion from the tissue were negligible. The uptake clearance of [11C]DPV was determined using eq. 1:
(1)where Xt, tissue is the amount of radioactivity as [11C]DPV in the liver or kidneys at time t, as determined by PET imaging, and Ct, blood is the concentration of radioactivity as [11C]DPV in the blood at time t, as determined by γ-counter measurement of the radioactivity in the blood taken from the femoral artery. The tissue uptake clearance [hepatic uptake clearance (CLuptake, liver) and renal uptake clearance (CLuptake, kidney)] was obtained from the initial slope of the plot of Xt, tissue/Ct, blood versus the AUC0-t, blood/Ct, blood, where AUC0-t, blood is the area under the concentration-time curve (AUC) for blood (AUCblood) from time 0 to time t. VE, tissue represents the initial distribution volume in the liver or kidneys at time 0, calculated from the y-intercept of the plot.
The canalicular efflux clearance with regard to the liver radioactivity (CLint, bile) was estimated using the following equation by noninvasive measurements of the radioactivity in the gallbladder and bile duct (the radioactivity excreted into the bile):
(2)where Xt, bile is the amount of radioactivity in the sum of the amounts of radioactivity in the gallbladder and bile duct at time t, as determined by PET imaging. In the control phase, significant radioactivity was also detected in the small intestines of volunteers 1 and 3, who did not show any radioactivity in the small intestine in the rifampicin-treated phase. We speculated that the radioactivity (1% and 18% of Xt, bile, respectively) originated from the bile and, thus, was added to Xt, bile values in the analysis. AUC0-t, liver is the AUC for liver from time 0 to time t. The CLint, bile value can be obtained from the slope of the plot of Xt, bile versus AUC0-t, liver. VE was calculated as the y-intercept of the integration plot.
Calculations for the In Vitro Kinetic Parameters in the Uptake Study Using Transporter-Expressing Cells.
The concentration dependence on the uptake velocity of DPV by OATP1B1 and OATP1B3 was fitted to the Michaelis-Menten equation, as follows:
(3)where ν is the uptake rate of the substrate (pmol/min per milligram of protein), S is the substrate concentration in the medium (μM), Km is the Michaelis constant (μM), Vmax is the maximum uptake rate (pmol/min per milligram protein), and Pdif is the clearance for nonsaturable component (ml/min per milligram protein). For OATP1B1, S was not included in the parameter estimation. In the parameter estimation of Km and Vmax values for OATP1B3, Pdif was fixed as the uptake clearance at the highest concentration, 0.433 μl/min per milligram of protein. This uptake value was identical to the uptake clearance of DPV in the empty vector–transfected cells.
Statistical Analyses.
Data were calculated as the mean ± S.D. for six determinations. Student's two-tailed paired t test was used to identify significant differences between the control group and the rifampicin-treated group. A two-way analysis of variance with the Bonferroni multiple comparisons test was used to assess the effect of rifampicin on the time profiles of tissue radioactivity. Statistical significance was set at P < 0.05.
Biodistribution of Radioactivity in the Human Abdominal Region and Blood After the Intravenous Administration of [11C]DPV.
Representative PET images of [11C]DPV in abdominal tissues with and without rifampicin treatment in the healthy subjects are shown in Fig. 2. Representative PET images of [11C]DPV in abdominal tissues without rifampicin treatment in the healthy subjects and the DJS patient are shown in Fig. 7. The distribution of [11C]DPV was observed in the liver and kidney and was negligible in the other tissues (Supplemental Fig. 1).
Color-coded PET images of the abdominal region of healthy male subjects after the administration of [11C]DPV with and without rifampicin. Coronal maximum-intensity projection PET images of the radioactivity in the abdominal region were captured at 2, 4, 10, and 60 minutes after the administration of [11C]DPV (84 ± 11 MBq/body, 6.0 ± 4.0 nmol/body) in the absence (A) or presence (B) of 600 mg of rifampicin. SUV, standardized uptake value.
The time profiles of the mean total tissue and blood radioactivity and AUCblood are shown in Fig. 3 and Table 1. Radioactivity peaked at 0.0237% ± 0.003% of the dose in the livers of control subjects at 13 minutes after administration and 0.0080% ± 0.0013% of the dose in the liver of rifampicin-treated subjects at 1.9 minutes after administration. Rifampicin treatment significantly reduced the amount of radioactivity excreted into the bile (1.5% ± 1.0% of the dose in rifampicin-treated subjects versus 11.2% ± 2.7% of the dose in control subjects), and delayed the elimination of radioactivity from the systemic circulation (Fig. 3). Consequently, the AUCblood values in rifampicin-treated subjects were 1.7-fold higher than those in control subjects (0.180% ± 0.061% versus 0.103% ± 0.020% of the dose⋅minutes per milliliter) (Table 1). In contrast, rifampicin treatment slightly delayed the elimination of radioactivity from the kidney, resulting in an increase in the AUC for the kidney (Table 1).
Radioactivity-time profiles in the blood, liver, and kidneys of healthy male subjects after the intravenous administration of [11C]DPV. Time profiles of the mean ± S.D. (n = 6) radioactivity in the blood (A), liver (B), gallbladder and bile duct (C), and kidney (D) were determined. PET imaging of the abdominal region and blood sampling were performed over 60 minutes after the administration of [11C]DPV to subjects who received (closed circles) or did not receive (open circles) rifampicin pretreatment.
Pharmacokinetic parameters of [11C]DPV after intravenous administration in control and rifampicin subjects
CLuptake, liver, CLuptake, kidneys, VE, liver, and VE, kidneys were calculated from eq. 1, and CLint, bile was obtained from eq. 2. Values are the mean ± S.D. (n = 6), unless otherwise indicated. The unit of clearance determined by the integration plot analysis was converted using the body weight, liver weight, and kidney weight in Supplemental Table 1.
The time profiles of the mean total tissue and blood radioactivity in the healthy subjects and the DJS patient are shown in Figs. 3 and 8. Radioactivity peaked at 0.0237% ± 0.003% of the dose in the livers of healthy subjects at 13 minutes after administration, and at 0.0164% of the dose in the liver of DJS patient at 17 minutes after administration. The amount of radioactivity in the DJS patient excreted into the bile was less than that in healthy subjects (3.88% of the dose in the DJS patient versus 11.2% ± 2.7% of the dose in healthy subjects) (Fig. 8). The AUCblood values in the DJS patient were slightly higher than those in healthy subjects (0.137% versus 0.103% ± 0.020% of the dose⋅minutes per milliliter).
Radiometabolite Analyses of [11C]DPV in the Blood by TLC Autoradiography.
Figure 4 shows representative TLC autoradiograms of extracts from the blood by 30 minutes after the administration of [11C]DPV to the control and rifampicin-treated subjects. A TLC analysis revealed one identifiable metabolite (M1) with [11C]DPV in the blood in all groups. The abundance of parent [11C]DPV in the blood (percentage of the total activity applied) in the control subjects (n = 6) was 100% at 2 and 5 minutes, and decreased with time, as follows: 87% ± 17% at 10 minutes, 70% ± 13% at 20 minutes, and 63% ± 16% at 30 minutes. The abundance of parent [11C]DPV in the blood of rifampicin-treated subjects (n = 6) showed profiles similar to those of the control subjects, as follows: 100% at 2 minutes, 98% ± 4% at 5 minutes, 97% ± 4% at 10 minutes, 85% ± 14% at 20 minutes, and 86% ± 14% at 30 minutes, respectively. Overall, the TLC assay with blood samples suggests that similar parent [11C]DPV profiles in the blood were exhibited by all groups and that the treatment by rifampicin was likely to have negligible effects on the metabolism of [11C]DPV.
Representative radiochromatograms of [11C]DPV and its metabolites in blood. Blood specimens collected from control and rifampicin-treated subjects at 2, 5, 10, 20, and 30 minutes were subjected to TLC and then analyzed by autoradiography. Au-std, [11C]DPV as an authentic standard.
The Liver Uptake and Canalicular Efflux Clearance of [11C]DPV.
Integration plots were drawn to calculate the tissue uptake clearance of radioactivity (Figs. 5 and 8). The slopes of the plots were determined across the linear range. CLuptake, liver values in the control subjects ranged from 32% to 76% of the hepatic blood flow rate (21 ml/min per kilogram) (Davies and Morris, 1993). Rifampicin treatment significantly decreased the CLuptake, liver value (by 58% ± 19%; P < 0.01) compared with that in control subjects, but had no effect on CLuptake, kidney, which was 7.83 ± 3.26 and 5.72 ± 3.80 ml/min per kilogram, respectively, in control and rifampicin-treated subjects.
An integration plot analysis after the intravenous administration of [11C]DPV. Integration plots were determined for the hepatic uptake (A), canalicular efflux (B), and renal uptake (C) clearance of [11C]DPV in the control and rifampicin-treated (600 mg/body) subjects. The hepatic uptake (D), canalicular efflux (E), and renal uptake (F) clearance were compared between the control and rifampicin-treated groups. The statistical significance was tested by Student's two-tailed paired t test (#P < 0.05; *P < 0.01).
Integration plots were also performed to determine the CLint, bile values of radioactivity across the linear range. Rifampicin treatment resulted in significant 40% lower CLint, bile values (0.129 ± 0.091 versus 0.221 ± 0.083 ml/min per kilogram, respectively) in rifampicin-treated subjects than in control subjects (Table 1), although its inhibitory effect varied from 8.6% to 87% depending on the volunteers. More than 30% reduction was observed in four of six subjects. Further, the CLint, bile values in the DJS patient was lower than that in the healthy subjects (0.102 versus 0.221 ± 0.083 ml/min per kilogram, respectively).
The Uptake of DPV by OATP1B1- and OATP1B3-Expressing HEK293 Cells.
The activities of OATP1B1 and OATP1B3 in HEK293 cells were confirmed by examining the uptake of [3H]E217βG, a prototypical substrate of OATPs, in the presence and absence of rifampicin (100 μM). The uptake of [3H]E217βG in the OATP1B1- and OATP1B3-expressing cells was significantly decreased in the presence of rifampicin (Supplemental Fig. 2). DPV (1 and 3 μM, respectively, for OATP1B1 and OATP1B3 tests) was significantly accumulated in both OATP1B1- and OATP1B3-expressing cells but was diminished in the presence of rifampicin (10 μM) (Fig. 6). The uptake rate of DPV in OATP1B1- and OATP1B3-expressing cells was determined at various substrate concentrations (3–300 μM) (Fig. 6). The uptake velocity of DPV by OATP1B1 and OATP1B3 follows the Michaelis-Menten equation. The Km (micromolar) and Vmax values of DPV were 6.52 ± 2.16 and 42.1 ± 3.2 pmol/min per milligram of protein for OATP1B1, and 17.1 ± 8.3 and 39.6 ± 12.1 pmol/min per milligram of protein for OATP1B3, respectively.
The uptake of DPV by OATP1B1- and OATP1B3-expressing HEK293 cells. (A) The uptake of DPV (1 and 3 μM, respectively, for OATP1B1 and OATP1B3 tests) by empty vector–transfected cells (square symbol), OATP1B1-expressing cells (circle symbol), and OATP1B3-expressing cells (diamond symbol) in the absence (open symbol) and presence (closed symbol) of rifampicin (10 µM) was determined at 37°C at the designated time points. (B) The concentration dependence of the uptake is shown as a Michaelis-Menten plot and Eadie-Hofstee plot (inset). The uptake of DPV for 2 minutes was determined at various substrate concentrations (1–100 μM for OATP1B1, and 3–300 μM for OATP1B3). Solid lines represent the fitted lines. Details of the fitting are described in Materials and Methods. Each point represents the mean ± S.E. (n = 4 for OATP1B1; n = 3 for OATP1B3).
We demonstrated that [11C]DPV, a newly developed PET probe, was a useful novel noninvasive method of assessing the activity of in vivo drug transporters in humans. We showed that PET imaging with [11C]DPV enabled the quantitative evaluation of the function of OATP1Bs in hepatic uptake, and of MRP2 in canalicular efflux. This helped to clarify the drug transporter–mediated DDIs.
Hepatic and/or renal elimination is the major pathway of most drugs in a living body. Since multispecific drug transporters, such as OATP1Bs and organic anion transporters (OATs), are indispensable in the hepatic and renal uptake of various drugs, especially those with anionic properties, the accurate functional characterization of these transporters is essential to drug development. Noninvasive PET imaging methods potentially offer new approaches to the direct estimation of the functional activity of transporters that contribute to drug disposition, even in humans. Indeed, through their PET imaging–based pharmacokinetic analysis, Takashima et al. (2010, 2012) demonstrated that 15R-[11C]TIC was an OATP1Bs transporter substrate in rat and human livers and directly estimated the pharmacokinetic parameters of CLuptake, liver. The CLuptake, liver in humans was one-third of that in rats. The magnitude of the relationship between CLuptake, liver and the hepatic blood flow rate showed a clear species difference; the CLuptake, liver in rats was comparable to the hepatic blood flow rate (55 ml/min per kilogram), whereas that in humans was below the hepatic blood flow rate (Takashima et al., 2012). A large amount of the multiple metabolites (rats, three metabolites; humans, four metabolites) of 15R-[11C]TIC-Me in liver might have hampered the precise evaluation of the hepatobiliary clearance in vivo (Takashima et al., 2010, 2012).
We found that the distribution of the pravastatin analog [11C]DPV is limited to the liver and kidney in humans, as previously reported in rats (Shingaki et al., 2013). Rifampicin treatment markedly decreased the liver radioactivity, and its AUC, and reduced the cumulative radioactivity of [11C]DPV associated with the gallbladder and bile duct (Fig. 3; Table 1). Consequently, rifampicin significantly delayed the elimination of radioactivity from the blood (Fig. 3; Table 1). Although rifampicin treatment did not affect the maximum radioactivity in the kidney, a slight delay caused by prolonged systemic exposure was observed. These changes were in good agreement with the clinical studies of 15R-[11C]TIC (Takashima et al., 2012), suggesting that hepatic elimination accounts for a significant fraction of the total elimination pathway of [11C]DPV from the blood circulation.
The tissue uptake clearances (CLuptake, liver and CLuptake, kidney), regarding the blood concentrations, and intrinsic canalicular efflux (CLint, bile), regarding the liver concentrations, were determined by an integration plot analysis. Rats exhibited a blood flow–limited liver uptake of [11C]DPV (Shingaki et al., 2013). Meanwhile, the CLuptake, liver was half of the human hepatic blood flow rate (20.7 ml/min per kilogram) (Table 1). CLuptake, liver values in humans (544 ± 204 µl/min per gram of liver) were 3-fold lower than the previously reported corresponding parameters in rats (1800 µl/min per gram of liver). Considering the blood flow–limited liver uptake in rats, the species difference in the intrinsic tissue uptake clearances will be more remarkable. The CLuptake, liver value of [11C]DPV was almost equivalent to the nonrenal clearance (7.2 ml/min per kilogram) of pravastatin in humans (Singhvi et al., 1990). This is consistent with in vitro data, where the transport activities of DPV and pravastatin by OATP1B1 and OATP1B3 were similar (Supplemental Fig. 3). Further, the Km value for OATP1B1 was almost the same as that of pravastatin, as reported previously (27, 29, and 35 μM, respectively) (Hsiang et al., 1999; Sharma et al., 2012; Izumi et al., 2015). More importantly, significant reduction in the CLuptake, liver of [11C]DPV by rifampicin administration supports (Fig. 5) that this PET tracer is applicable for the quantitative evaluation of the human hepatic uptake mediated by OATP1B1 and OATP1B3. Meanwhile, the modest effect of rifampicin (Table 1) suggests that it is necessary to validate the dynamic range for the analysis.
Although a PET imaging–based pharmacokinetic analysis allows for the quantitative evaluation of CLint, bile, it is limited by its inability to distinguish between the radioactivity of the parent drug and its metabolites in the targeted tissue. [11C]DPV is hardly metabolized in in vitro human hepatocytes (Shingaki et al., 2013), whereas our radiometabolite analysis in humans confirmed the presence of a metabolite (M1) with radioactivity in the blood. The abundance of the M1 in the control subjects (n = 6) increased with time and accounted for approximately 40% of the total activity at 30 minutes after administration. Since one metabolite predominated in the blood 20 minutes after the injection of 15R-[11C]TIC-Me (Takashima et al., 2012), [11C]DPV is a more metabolically stable PET probe than 15R-[11C]TIC-Me. However, the impact of metabolite formation on the evaluation of CLint, bile values of [11C]DPV remains unknown. Further studies are necessary to identify M1, and to clarify the MRP2 activities of the M1 in comparison with DPV to increase the certainty of CLint, bile measurement. Concurrently, we continue our efforts to develop a nonmetabolizable PET probe for OATP1Bs and MRP2.
The functional activity of Mrp2 in rats was evaluated based on a [11C]DPV PET imaging analysis. Rifampicin-treated and Mrp2-defective rats showed decreased CLint, bile (Shingaki et al., 2013). Large differences are seen in the liver MRP2 activity of rats and other species, including humans (Ishizuka et al., 1999; Niinuma et al., 1999). The ATP-dependent uptake of pravastatin by the canalicular membrane vesicles in humans was six times lower than that in rats (Niinuma et al., 1999). Consistently, the CLint, bile of [11C]DPV (10.2 µl/min per gram of liver) in control subjects was 30 times lower than those in rats (298 µl/min per gram of liver) (Shingaki et al., 2013). The effect of rifampicin on the CLint, bile was statistically significant, but varied; the value decreased by >30% in four of six subjects, whereas the others showed a reduction of ≤10%. To strengthen the application of [11C]DPV as a PET probe for MRP2, an additional clinical study was conducted in a patient with DJS caused by hereditary MRP2 deficiency. The CLint, bile in the DJS patient (0.102 ml/min per kilogram) was smaller than that in control subjects (0.221 ml/min per kilogram) (Figs. 7 and 8), supporting the importance of MRP2 in the transport process. The impact of genetic polymorphisms of OATP1Bs and MRP2 on the pharmacokinetic parameters remains to be clarified by [11C]DPV PET imaging, because the pharmacokinetics of pravastatin showed large interindividual variation due to genetic polymorphisms of not only OATP1Bs but also MRP2 (Ieiri et al., 2009).
Color-coded PET images of the abdominal region of healthy male subjects (A) and a DJS patient (B) after the administration of [11C]DPV. Coronal maximum-intensity projection PET images of the radioactivity in the abdominal region were captured at 5, 20, 30, and 60 minutes after the administration of [11C]DPV (82 ± 12 MBq/body, 8.7 ± 5.0 nmol/body). SUV, standardized uptake value.
Radioactivity-time profiles of the blood, liver, and gallbladder and bile duct, and integration plot analysis of the gallbladders and bile ducts of healthy male subjects and a DJS patient after the intravenous administration of [11C]DPV. The time profile of the mean ± S.D. radioactivity in the blood (A), liver (B), and gallbladder and bile duct (C) of control subjects (n = 6) was determined. The time profile of the DJS patient was the data from a single patient. PET imaging of the abdominal region and blood sampling were performed over 60 minutes after the administration of [11C]DPV to the control subjects (open circles) or the DJS patient (closed diamonds). (D) Integration plots were determined for the canalicular efflux clearance of [11C]DPV in the control subjects and the DJS patient. Closed circles represent the control subjects. Closed diamonds represent the DJS patient.
Since DPV is efficiently transported by OAT3, but not by OAT1 (Supplemental Fig. 4), this transporter will mediate the basolateral uptake in the kidney, as suggested for pravastatin (Watanabe et al., 2011). CLuptake, kidney was decreased in four of six subjects by rifampicin treatment (Fig. 5). The factor accounting for the interindividual variability in the effect of rifampicin remains unknown. Such interindividual variability was also observed to a lesser extent in the renal clearance of pravastatin, which was decreased in 3 of 10 subjects (Kyrklund et al., 2004). Provided that rifampicin is a weak inhibitor of OAT3 (Kusuhara et al., 2013), the inhibition of OAT3-mediated uptake is an unlikely mechanism.
This study highlighted the usefulness of PET imaging in investigating pharmacokinetics and DDIs, particularly in the tissue concentrations caused by the inhibition of the transporter function. The tissue concentration of drugs is associated with the drug response when an intracellular protein is the target. However, when the uptake involves transporters, the prediction of the tissue concentration under both control and DDI conditions must be further clarified. A clinical PET study could determine the tissue concentration when the drug is labeled by a positron-emitted isotope. Furthermore, pharmacokinetic analyses of the PET data will help to clarify the DDI mechanisms, including some that may have been overlooked. Indeed, the present study suggests that the DDI mechanisms of rifampicin involve the inhibition of the canalicular efflux in some patients, as well as the sinusoidal uptake by OATP1B1/OATP1B3. There are two factors accounting for interindividual variability in CLint, bile to be investigated: the interindividual difference in MRP2 sensitivity to rifampicin caused by its genetic polymorphisms; and that in the unbound liver concentration of rifampicin caused by interindividual difference in the membrane transport and metabolism. Moreover, the accumulation of data on the in vivo intrinsic parameters of the elements across the drugs will provide correlations to in vitro parameters and help identify the empirical scaling factors that may improve the precision of the in vitro-in vivo extrapolation to new chemical entities. Molecular imaging probes for the quantitative functional characterization of drug transporters are still being developed by our group and others [[11C]rosuvastatin, Oatps, Bcrp (breast cancer resistance protein), and Mrp2 in rats] (He et al., 2014), but the characterization of drug transporters and DDIs in humans has not yet been fully achieved. Further enrichment of these probes is necessary to increase the predictability of these transporters.
In conclusion, our findings demonstrated that noninvasive PET imaging with [11C]DPV as a PET probe with fewer metabolites than 15R-[11C]TIC-Me can assess the hepatobiliary transport by OATP1Bs and MRP2. Therefore, [11C]DPV is a useful probe for the kinetic analysis of hepatobiliary transport in humans.
The authors thank the investigators and subjects who participated in this study for valuable help and cooperation; as well as Tomotaka Shingaki, formerly of CLST, for clinical support. We also thank Masashi Okamoto and Hisako Kobata (Osaka City University Hospital) for expert technical assistance and supervision of this study, and Ryo Fujino and Shunske Aoyama (ADME & Toxicology Research Institute, Sekisui Medical Co., Ltd., Ibaraki, Japan) for contributing to in vitro experiments using HEK293 cells expressing OATP1B1.
Authorship Contributions
Participated in research design: Kaneko, Maeda, Enomoto, Kawamura, Kawada, Kusuhara, Sugiyama, Watanabe.
Conducted experiments: Tanaka, Ishii, Katayama, Kawahata, Yamanaga, Wada, Miyake.
Contributed new reagents or analytic tools: Tanaka, Katayama, Kawahata, Yamanaga, Wada, Kawabe, Shiomi.
Performed the data analysis: Kaneko, Katayama, Nakaoka, Irie, Miyake, Toshimoto.
Wrote or contributed to the writing of the manuscript: Kaneko, Maeda, Cui, Kusuhara, Sugiyama, Watanabe.
Accepted March 13, 2018.
This work was supported in part by a consignment expense from the Molecular Imaging Program on "Research Base for Exploring New Drugs" from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (S) [Number 24229002]. T.S. is a former employee of RIKEN Center for Life Science Technologies (CLST) and has authored several papers while at CLST.
https://doi.org/10.1124/dmd.118.080408.
This article has supplemental material available at dmd.aspetjournals.org.
area under the concentration-time curve
AUC0-t
blood, area under the concentration-time curve for blood from time 0 to time t
liver, area under the concentration-time curve for liver from time 0 to time t
[11C]DPV
sodium (3R, 5R)-3, 5-dihydroxy-7-((1S, 2S, 6S, 8S)-6-hydroxy-2-methyl-8-((1-[11C]-(E)-2-methyl-but-2-enoyl) oxy)-1, 2, 6, 7, 8, 8a-hexahydronaphthalen-1-yl) heptanoate
AUCblood
area under the concentration-time curve for blood
bile, canalicular efflux clearance
CLuptake
kidney, renal uptake clearance
liver, hepatic uptake clearance
blood, concentration of radioactivity as [11C]DPV in the blood at time t
drug-drug interaction
Dubin-Johnson syndrome
E217βG
estradiol 17β glucuronide
Michaelis constant
MRP2/Mrp2
multidrug resistance–associated protein 2
organic anion transporter
OATP/Oatp
organic anion–transporting polypeptide
Pdif
clearance for passive diffusion
15R-[11C]TIC-Me
(15R)-16-m-[11C]tolyl-17, 18, 19, 20-tetranorisocarbacyclin methyl ester
bile, amount of radioactivity in the sum of the amounts of radioactivity in the gallbladder and bile duct at time t
tissue, amount of radioactivity as [11C]DPV in the liver or kidneys at time t
Copyright © 2018 by The Author(s)
This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.
Davies B and
Morris T
(1993) Physiological parameters in laboratory animals and humans. Pharm Res 10:1093–1095.
Dubin IN and
Johnson FB
(1954) Chronic idiopathic jaundice with unidentified pigment in liver cells; a new clinicopathologic entity with a report of 12 cases. Medicine (Baltimore) 33:155–197.
He J,
Yu Y,
Prasad B,
Link J,
Miyaoka RS,
Chen X, and
Unadkat JD
(2014) PET imaging of Oatp-mediated hepatobiliary transport of [(11)C] rosuvastatin in the rat. Mol Pharm 11:2745–2754.
Hirano M,
Shitara Y, and
Sugiyama Y
(2004) Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans. J Pharmacol Exp Ther 311:139–146.
(2006) Drug-drug interaction between pitavastatin and various drugs via OATP1B1. Drug Metab Dispos 34:1229–1236.
Hsiang B,
Zhu Y,
Wang Z,
Wu Y,
Sasseville V,
Yang WP, and
Kirchgessner TG
(1999) A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liver-specific human organic anion transporting polypeptide and identification of rat and human hydroxymethylglutaryl-CoA reductase inhibitor transporters. J Biol Chem 274:37161–37168.
Ieiri I,
Higuchi S, and
(2009) Genetic polymorphisms of uptake (OATP1B1, 1B3) and efflux (MRP2, BCRP) transporters: implications for inter-individual differences in the pharmacokinetics and pharmacodynamics of statins and other clinically relevant drugs. Expert Opin Drug Metab Toxicol 5:703–729.
Ijuin R,
Takashima T,
Watanabe Y,
Sugiyama Y, and
Suzuki M
(2012) Synthesis of [(11)C]dehydropravastatin, a PET probe potentially useful for studying OATP1B1 and MRP2 transporters in the liver. Bioorg Med Chem 20:3703–3709.
Ishizuka H,
Konno K,
Shiina T,
Naganuma H,
Ito K,
Suzuki H, and
(1999) Species differences in the transport activity for organic anions across the bile canalicular membrane. J Pharmacol Exp Ther 290:1324–1330.
Izumi S,
Nozaki Y,
Komori T,
Takenaka O,
Kusuhara H, and
(2015) Investigation of the impact of substrate selection on in vitro organic anion transporting polypeptide 1B1 inhibition profiles for the prediction of drug-drug interactions. Drug Metab Dispos 43:235–247.
Kim DC,
Satoh H,
Fuwa T,
Iga T, and
Hanano M
(1988) Kinetic analysis of in vivo receptor-dependent binding of human epidermal growth factor by rat tissues. J Pharm Sci 77:200–207.
Kitamura S,
Wang Y, and
(2008) Involvement of multiple transporters in the hepatobiliary transport of rosuvastatin. Drug Metab Dispos 36:2014–2023.
Kusuhara H
(2013) Imaging in the study of membrane transporters. Clin Pharmacol Ther 94:33–36.
Kusuhara H,
Miura M,
Yasui-Furukori N,
Yoshida K,
Akamine Y,
Yokochi M,
Fukizawa S,
Ikejiri K,
Kanamitsu K,
Uno T, et al.
(2013) Effect of coadministration of single and multiple doses of rifampicin on the pharmacokinetics of fexofenadine enantiomers in healthy subjects. Drug Metab Dispos 41:206–213.
Kyrklund C,
Backman JT,
Neuvonen M, and
Neuvonen PJ
(2004) Effect of rifampicin on pravastatin pharmacokinetics in healthy subjects. Br J Clin Pharmacol 57:181–187.
Lee JH,
Chen HL,
Ni YH,
Hsu HY, and
Chang MH
(2006) Neonatal Dubin-Johnson syndrome: long-term follow-up and MRP2 mutations study. Pediatr Res 59:584–589.
Maeda K
(2015) Organic anion transporting polypeptide (OATP)1B1 and OATP1B3 as important regulators of the pharmacokinetics of substrate drugs. Biol Pharm Bull 38:155–168.
Maeda K and
(2008) Impact of genetic polymorphisms of transporters on the pharmacokinetic, pharmacodynamic and toxicological properties of anionic drugs. Drug Metab Pharmacokinet 23:223–235.
Matsushima S,
Kondo C,
Sasaki M,
(2005) Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein. J Pharmacol Exp Ther 314:1059–1067.
Nies AT and
Keppler D
(2007) The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch 453:643–659.
Niinuma K,
Kato Y,
Suzuki H,
Tyson CA,
Weizer V,
Dabbs JE,
Froehlich R,
Green CE, and
(1999) Primary active transport of organic anions on bile canalicular membrane in humans. Am J Physiol 276:G1153–G1164.
Sharma P,
Butters CJ,
Smith V,
Elsby R, and
Surry D
(2012) Prediction of the in vivo OATP1B1-mediated drug-drug interaction potential of an investigational drug against a range of statins. Eur J Pharm Sci 47:244–255.
Shingaki T,
Onoue T,
Katayama Y,
Okauchi T,
Hayashinaka E,
Cui Y,
Wada Y, et al.
(2013) Evaluation of Oatp and Mrp2 activities in hepatobiliary excretion using newly developed positron emission tomography tracer [11C]dehydropravastatin in rats. J Pharmacol Exp Ther 347:193–202.
Shitara Y,
Sato H, and
(2005) Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs. Annu Rev Pharmacol Toxicol 45:689–723.
Singhvi SM,
Pan HY,
Morrison RA, and
Willard DA
(1990) Disposition of pravastatin sodium, a tissue-selective HMG-CoA reductase inhibitor, in healthy subjects. Br J Clin Pharmacol 29:239–243.
Hashizume Y,
Murai M,
Wada Y,
Watanabe Y
(2011) The involvement of organic anion transporting polypeptide in the hepatic uptake of telmisartan in rats: PET studies with [11C]telmisartan. Mol Pharm 8:1789–1798.
Shigihara Y,
Ishii H,
Shiomi S,
Nakae T,
Watanabe Y, et al.
(2012) PET imaging-based evaluation of hepatobiliary transport in humans with (15R)-11C-TIC-Me. J Nucl Med 53:741–748.
Doi H,
Suzuki M,
Kusuhara H, et al.
(2010) Positron emission tomography studies using (15R)-16-m-[11C]tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester for the evaluation of hepatobiliary transport. J Pharmacol Exp Ther 335:314–323.
Wu C,
Takashima-Hirano M,
(2013) Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with 11C-SC-62807. J Nucl Med 54:267–276.
van Dijk R,
Beuers U, and
Bosma PJ
(2015) Gene replacement therapy for genetic hepatocellular jaundice. Clin Rev Allergy Immunol 48:243–253.
Watanabe T,
(2010) Application of physiologically based pharmacokinetic modeling and clearance concept to drugs showing transporter-mediated distribution and clearance in humans. J Pharmacokinet Pharmacodyn 37:575–590.
Debori Y,
Kondo T,
Nakayama H,
Horita S,
Ogilvie BW,
Parkinson A, et al.
(2011) Prediction of the overall renal tubular secretion and hepatic clearance of anionic drugs and a renal drug-drug interaction involving organic anion transporter 3 in humans by in vitro uptake experiments. Drug Metab Dispos 39:1031–1038.
Willmann JK,
van Bruggen N,
Dinkelborg LM, and
Gambhir SS
(2008) Molecular imaging in drug development. Nat Rev Drug Discov 7:591–607.
Maeda K, and
(2013) Hepatic and intestinal drug transporters: prediction of pharmacokinetic effects caused by drug-drug interactions and genetic polymorphisms. Annu Rev Pharmacol Toxicol 53:581–612.
Thank you for sharing this Drug Metabolism & Disposition article.
You are going to email the following A Clinical Quantitative Evaluation of Hepatobiliary Transport of [11C]Dehydropravastatin in Humans Using Positron Emission Tomography
Message Subject (Your Name) has forwarded a page to you from Drug Metabolism & Disposition
Message Body (Your Name) thought you would be interested in this article in Drug Metabolism & Disposition.
Human PET Study on Functional Analysis of OATP1Bs and MRP2
Drug Metabolism and Disposition May 1, 2018, 46 (5) 719-728; DOI: https://doi.org/10.1124/dmd.118.080408
DNA Methylation Regulates MRP3 Expression in Human Skin
MRP2 Inhibition by HIV Protease Inhibitors
Kinases and Protein-Interactions Impact on ABC Transporters
Show more Special Section on Transporters in Drug Disposition and Pharmacokinetic Prediction


text:
Virus é uma banda de rock argentina, fundamental dentro do estilo musical new wave da década de 1980 na América do Sul, liderada por Federico Moura até sua morte em dezembro de 1988, causada pelo HIV. Seu irmão tomou então o posto de vocalista principal e a banda continuou até o final de 1989.

Em março de 1994, a banda voltou a reunir-se para retomar suas atividades, continuando até os dias atuais, porém sem recuperar sua popularidade prévia.

Discografia
Wadu Wadu, (1981)
Recrudece, (1982)
Agujero interior, (1983)
Relax, (1984)
Locura, (1985)
Vivo volumen 2, (1986)
Virus vivo, (1986)
Grandes éxitos, (1987)
Superficies de placer, (1987)
Tierra del fuego, (1989)
Nueve, (1998)
América fatal (simple), (1998)
Obras cumbres, (2000)
Caja negra, (2006)

Ligações externas
Biografia da banda (Em espanhol)

Bandas da Argentina
Bandas de rock
Rock da Argentina
Bandas formadas em 1981


All content should map to a strategic plan. If you don't already have one, I can work with you to create a simple, actionable plan to help you choose a destination and route to get there, as well as activities to help you get the most out of your journey. This initial work can save time and money in the long run and improve results.
What's the difference? Copywriting is crafted to sell or promote something, whereas content writing is all about adding value for your readers so they'll want to engage with your brand. The two work together and sometimes overlap. Writing with a specific goal in mind makes it easier to measure its effectiveness. I can help you choose the right key performance indicators (KPIs) to support continuous improvement.
Perhaps an employee has gone on leave, or your business is in a rapid-growth phase. I can take a project off your plate or step in temporarily to manage a project, event, or team. I tend to prefer an agile approach to project management but can follow the methodology that works best for you to manage the specific projects at hand.


This very haunted, privately owned, mansion can be found at 476 Summit Avenue, in Saint Paul, Minnesota, on a street that showcases other "grand," old Victorian mansions, built around 1883 or thereabouts.
The Griggs Mansion has the dubious reputation of being the most haunted house in Saint Paul. This huge, 4 story, Victorian, well-kept sandstone mansion has 24 rooms, described as "cavernous," because of their high ceilings. Each floor has its landing, and a staircase leading up to the next floor. Dark, beautiful woodwork can be found throughout the mansion. There is a large room, just off the 4th floor landing, that has a beautiful, vast skylight, that was installed in 1939, when the mansion was being used as an art school. Behind the mansion there is a comfortable carriage house. Large trees around the mansion provide shade, and a sandstone wall that surrounds the mansion and its grounds provides privacy. It is privately owned by people who don't want to be bothered with ghost hunters, so please respect their privacy.
In 1883, a wealthy wholesale grocery businessman, Chauncey Griggs, built this lovely mansion, but lived in it for only four years, before moving onto greener pastures on the West coast, where he expanded his business interests to include the lumber and transportation markets. Over its 100+ years, the mansion has been a private residence, and at one time was divided into apartment units as well. Many families/ individuals, who moved in, spent a lot of money on upkeep, furnishings, servants, etc., but wound up leaving after only a few years.
Also, as mentioned above, the mansion became an art school in 1939, and stayed there for 25 years, until the new Arts and Sciences Center was built. Many students and teachers were glad that the new building was built, because now they didn't have to share their school with seen and unseen presences.
While a lot of "supernatural activity" seems to be centered around the 4th floor, many parts of the house have been visited by entities as well. Six or seven entities have been felt, heard, made their existence known by various means and have actually been seen throughout the years. The mansion's spiritual occupants are a young maid, a gardener, a child apparition, a thin man in a black suit, a teenager, Amy, and a Civil War general, perhaps Chauncey Griggs himself. The 7th sighting could've been any of the male apparitions, because only the head of an unclear, male apparition appeared.
1) One of the most strongly felt presences is that of a young maid, who in 1915, hung herself off the 4th floor landing, suffering from depression over an ended romance. Earlier in the 20th century, she appeared to a young servant and a butler in the 4th floor hallway. Her presence has been felt strongly by many people throughout the years. She perhaps is one of the entities that climbs the stairs to the fourth floor, to her unseen rope on the landing there. While climbing this staircase, and standing in the hall as well, people during the day have felt a "sense of foreboding," anxiety, and feelings of distress, that could be quite strong, as three newsmen found out one night, when they came to gather information for a series on ghosts. After setting up camp in the big room on the top floor, and armed with two cameras; one regular and one with infra-red aimed at the well-lighted, top of the stairs and 4th floor hall, and a tape recorder, the men each went out to the hall, but scooted back to the room when they were filled with an overwhelming sense of fear. They heard heavy footsteps coming up those stairs. One brave newsman went though the hall to the stairway, and though he saw nothing, he felt a strong presence on the stairs. They left in a hurried manner together down the stairs, feeling nothing was worth staying a night there.
2) The gardener - Charles Wade, who was an intense, fanatical worker, took great pride in keeping the gardens in magnificent shape, and would often use books in the mansion's well-stacked library to help him in his work. Various people have heard an unseen presence frantically, flipping through the books. Perhaps this distressed entity died before he was finished with his latest gardening project, and feels he has unfinished business.
3) The thin man, dressed in a black suit and top hat - This apparition visited people in many rooms of the mansion, but two eye-witness accounts are given below.
A) A Dr. Delmar Kolb in the early 1950's, joined the art school's teaching staff, and moved into the front basement apartment in the mansion. He awoke one night by the pressure of two dead, cold fingers on his forehead, and found himself in a cold sweat. After turning on the light, he saw a blue flash that disappeared. Two nights later, he was again awakened to find an clear, detailed apparition of a man dressed in a black suit and top hat, who was standing at the foot of his bed. At first, the alarmed Dr. Kolb thought it was a prowler dressed in costume, but realized it was a ghost when the apparition dissolved into the brick wall.
B) In 1964, a Mr.Weschke bought the mansion with the idea of using the place as both an office and his home. He published books on the occult. In 1967, Mr. Weschke was working at the desk in the library. When he stopped to take a break, he saw visually in the doorway of the library, a thin, long-faced man with white hair, dressed in a black suit, who was studying him. The apparition faded away after about 30 seconds.
4) A Child apparition. - In 1959, two college students moved into the front basement apartment. One night, something woke up one student, who usually was a sound sleeper. H e looked up and saw the head of a child floating above his bed.
5) A teenager, Amy, and a Civil War general, perhaps Chauncey Griggs. When a St. Paul spiritualist/medium. By the name of Roma Harris came to visit the mansion, He felt the "shadowy presence" of a teenage girl, who calls herself Amy. She had had many happy moments playing the mansion's piano. She died young, and perhaps hangs around the mansion where she was happy making music. Perhaps she lived during the art school days of the mansion. Roma also "saw" a Civil war officer, dressed in a general's blue uniform, with the gold trim. As the original owner, Chauncey Griggs, was a Civil War officer, it is theorized that this entity is Chauncey, who likes to keep an eye on his mansion. It also could be another earlier, former owner, who didn't want to leave his mansion.
6) Many unexplained, but typical incidents common in haunted houses have been reported, though no specific entity has been found to be responsible for the noises, annoying behavior that makes the living uncomfortable or befuddled. Windows that had been firmly closed, even nailed shut, have been found open in the morning. Footfalls have been heard traveling up and down the staircases. Shadowy presences have been seen. Unsuspecting students who rented the apartments, have been shaken awake, to find an undistinguished apparition head floating above their beds. Doors open and close by themselves. "Rasping coughs" have been heard coming from empty rooms. Light bulbs suddenly shatter, paper bags have been known to jump off the shelves, and jump across the floor. People in the mansion have felt presences walk by them. Some of the presences happen to appreciate art. While working in the art studio room, with the skylight on the top floor, students had felt presences peering over their shoulder to get a good look at their drawings in progress, perhaps studying with interest the student's efforts.
Just ask the St. Paul Pioneer Press. When investigating this widely known haunted house, they got first hand confirmation when they themselves came in contact with ghostly manifestations.
The Griggs Mansion was one of the first haunted house stories I compiled from a variety of sources in 1998. Never saw a picture of it, until our road trip of the summer of 2007. It is an absolutely gorgeous example of a sandstone, 1883 Victorian, which dominates the street. It's both very spooky and quite a work of art.


MomCoach: "Mommy, my tummy hurts!"
It doesn't take long for a child to learn that the words, "Mommy, my tummy hurts!" will produce instant action on the part of Mommy. If the kid is lucky, it will mean a day home from school, lounging around watching unlimited movies and being waited on by Mommy. If Mommy is wise, she'll think twice before assuming the worst.
Junior is thinking, "I don't want to go to school today. We're having a test. I have to sit by a GIRL. I'd rather play my video game. I think my tummy hurts."
Mommy is thinking, "Hmmmm, Junior ate a good breakfast. He looks and acts normal. Isn't he having a test today? I think we'll chance it and see what happens." And Junior is sent off to school, complaining all the way.
Mom, I think I have the plague!
Most likely, Junior will be fine once he's at school. It's possible that he DOES have a tummy ache, but probably caused by some level of anxiety, not illness. Your observation of his behavior is key. You can usually tell when your child is legitimately sick. If he seems ok, he probably IS ok.
My mother was a master at seeing through my attempts to stay home because I was "sick". If I managed to actually stay home, I had to stay in bed, no TV and only books to read. I couldn't get up to play after the other kids were home from school and only had simple, bland food to eat. It wasn't very enjoyable and I usually miraculously recovered enough to go to school the next day.
Poor Moms! We're the ones who need "sick days"!


Need a brick mason for your home? Mississippi Brick Masonry can do everything from minor repairs to major additions. Cracks, Chimneys, Mailboxes, Outdoor Grills or any brick work around your home.
Let us bid on your next project! We can handle everything from new construction to rebuilds. No job too large or small. Just give us a call today and let us submit a bid.


In 2013, I came to see Pujya Swamiji and asked him for a message to take back to the Argentine students. He said, "I am really satisfied that you have enough number of Arsha Vidya teachers in Argentina to keep you all busy with the study of Vedanta. Vedanta study never ends. It has to be kept going. You can never take for granted that 'I have already studied this'. You have to keep studying, then only you understand that what you thought you understood was not understood.


A great vintage metal wire basket with handle that makes an excellent decor piece, or usage piece.
This item mixes mod geometry with rustic.
Great as a fruit or picnic basket -- or as a towel and toiletries basket.
Condition: In good vintage condition with natural aging / silver patina for a rustic look! Handle extends and closes smoothly.


First Week in the Pool!
It is hard to believe that the first week of Dolphins is finally over. It has been a busy and exciting week with a fabulous opening day for the swim team, a productive first diving practice, some fun water-polo and an eager group of girls learning about synchro.
We hope your athletes are already having fun. There is more fun in store.
We will need an army of volunteers that day and a schedule for sign-up will be emailed soon.
More information will be available at the parent information meetings.
We will be conducting some parent-information meetings on the dates indicated below and we strongly encourage at least one parent from each family to attend. This is an opportunity to learn about our league, the Association of Lakeshore Pools, to learn about upcoming competitions, any special requests from coaches particular to the aquatics sport, information about volunteering, and a chance to meet members of the parents' committee. See you there!
Many of you have been asking about clothing. All the clothing orders that were done on registration night are being delivered to us in one large delivery and will be distributed en mass on T-SHIRT DAY. This day will be advised once we have the delivery date from our clothing supplier (hopefully we'll know this date at the information sessions!). This is a day that you won't want to miss: not only do we distribute the clothing but it is a chance to socialize with fellow team members, we will organize a team continental breakfast, we do team photos and this year we are planning an additional surprise…more details to follow!
There are more exciting activities to come and we look forward to sharing them with you. Check back to the blog and re-read previous posts in case you've forgotten something. This is where all the info and news of the club will be shared so it is worth taking the time to read it.


text:
Despite Recall, Popular Cereal Sickens People in 36 States
Debbie Ray
Debbie Ray Published: September 5, 2018
David McNew/Getty Images
Honey Smacks cereal was recalled in June, 2018, after people were sickened by salmonella after eating it. Despite the recall, some people are still finding it on shelves and eating it. Federal health officials say that the total number of illnesses is now at 130 cases. The individuals affected live in 36 different states. Fortunately, no deaths have been reported. The Food and Drug Administration reports the cereal is still for sale in some stores, and urges everyone to stay away from it.
kellogg honey smacks recall
from FDA.gov
From the FDA:
Kellogg is asking that people who purchased potentially affected product discard it and contact the company for a full refund. Consumers seeking more information, including images of these products, can visit kelloggs.com/honeysmacksrecall or call 1-800-962-1413 from Monday – Friday, from 9 a.m. to 6 p.m. ET as well as Saturday and Sunday from 10 a.m. – 4 p.m. ET.
More from the FDA HERE.


The lady that confessed to this deserves a medal! While the rest of us were probably desperately scraping our nostrils for mucus leftovers during our childhood, she must have indulged in proper, nutritious food - like macarons, for example.
Also - please send over any ideas of ridiculous life achievements and I will illustrate the best ones!
It's no secret: I can't swim. Just like I can't drive a car, ride a bike or do the things that generally make a good life.
However, during the summer of my 29th birthday, I managed to float. My summer ended with a glorious swimmer's ear. I didn't like water anyway!
This is something that only my fellow countrymen will get: owning a Dacia 1300 (willingly) is odd. But an achievement nontheless.
Having said that, I would still probably love to own a Trabant, but this is clearly one of my hipsterish phases.
birthday, when I realised that I haven't achieved much from the grown-ups point of view. But I wanted something to brag about anyway.
Lucky guys and their freakin' hair! This one's an illustration dedicated to all Johnny Bravos in the world.
I sort of feel for the bald sirs.
It's time for some hurtful truth! One of life's greatest wins is collecting a generous number of cats.
Sure, you've got your entire left to do that, but starting young builds experience and skill.
So for all those people feeling this, here's one for you: Achievement no 4 - Becoming a cat lady. Or sir. Whatevs.
To continue my 'On turning 30' series of ridiculous achievements, I thought I'd post one close to my heart: the food baby issue.
There you go, nice man on the tram giving up your seat for me: the truth has been spoken!
PS: I am still waiting for your list of silly achievements. You don't have to be 30 to join the discussion. I will pick the ones that inspire me and will illustrate them on my blog.
'On turning 30' is an illustrated series of infamous life wins that make you proud for some stupid reason. I have started this series on my 30th birthday, when I realised that I haven't achieved much from the grown-ups point of view. But I wanted something to brag about anyway.
So I turn 30 this week. Not sure what my plans are: either cry in my pyjamas eating Ben&Jerry's or shamelessly drink in celebration of it.
Either the turnout, thought I'd mark this glorious achievement (heck, 30 was the average life span in the 15th century! :D) with an illustrated series that celebrates my fulfilling life so far. So many important things to share!
I am planning on creating these achievements throughout the year and don't intend to keep them personal. So if you've got a ridiculous achievement that you'd like to share, send me a comment or a message and I will illustrate it and feature it in here.
Let the absurd plethora of achievements flow!
So I'll start with no 1: the fact that I managed to get (and keep alive so far) two Guinea pigs. I feel like such a responsible grown-up right now. I even turned the big one into this big mushy love-begging marshmallow: oinking for affection and cuddling with us on a blanket while watching movies. The little one is still an untamed beast.


text:
A Computable General Equilibrium Analysis of Energy Policies in Malaysia.
Idris, Jasmin. (2008) A Computable General Equilibrium Analysis of Energy Policies in Malaysia. Doctoral thesis, University of Surrey (United Kingdom)..
U500693.pdf
Malaysia has recently implemented a subsidy reduction policy on domestic fuels such as motor petrol, diesel and liquefied petroleum gas (LPG). In addition, in its five-year national plan (2005-2010), Malaysia also proposed a 350 megawatts (MW) electricity generation capacity from renewable energy technologies. In this thesis, a Computable General Equilibrium model (CGE) is used to measure the economic, welfare and the environmental impacts of the above policies. A model for tax and trade analysis for Malaysia (M2-TMAS) was developed to investigate the effects of alternative policies of recycling savings, due to subsidy reduction, back to the economy. M2-TMAS is a static, small open economy model with a data set from the recent 2000 input-output table. Results show that the adverse impact of fuel subsidy reduction could be mitigated through an appropriate savings recycling policy. A model for energy, economy and the environment for Malaysia (M3-EMAS) extends the model to include detailed representation of electricity sector, incorporating CO2 emissions resulting from the consumption of energy products. The model is used to analyse the proposed renewable energy portfolio in power generation. Results from this analysis suggest that the existing regulated natural gas price to power producers does not pose a barrier to the penetration of renewable energy technologies, although transferring those subsidies to renewable energy producers could encourage its share. Thus, if the regulated price is to be maintained, the portfolio target could be met through direct subsidies to renewable energy producers.
Idris, Jasmin.


text:
Redskins Sign CB Josh Wilson
By Steve Shoup Jul 27, 2011, 7:45pm EDT
Share All sharing options for: Redskins Sign CB Josh Wilson
The Redskins have apparently found Carlos Rogers replacement today as they inked former Maryland CB Josh Wilson to a 3 year $13.5 million deal. Wilson was a former 2nd round pick of the Seattle Seahawks, who was traded last season to the Baltimore Ravens for a 5th round draft pick.
Wilson had an up-and-down Seahawk career, but turned in a fantastic campaign with the Ravens last season. He was rated the 5th best overall corner according to Pro Football Focus, and his 46.9% completion percentage against, ranked 10th in the league.
Wilson profiles as a good number two corner and should be usually matched up against quicker/smaller wide receivers. Look for him to be moved inside to the slot position, when opponents go 3 wide. While there are some concerns about his size and his average performance before last year, I think he is is well worth the investment by the Redskins.
At just 26 years of age, Wilson is an absolute steal at $4.5 million a year, and should make Redskins fans quite happy with his ability to catch the football. He is considered a high character guy, with strong local ties and should be a quick fan favorite (also he isn't a Wide Receiver).