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Book reviews
Reviews By splashofourworlds.blogspot.com
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Hush, Hush
USD$15.66 - Save $4.33 21% off - RRP $19.99
- Published: 13 Oct 2009
- Format: Hardback
under review
Hush Hush!
written: 08 Sep 2010
When I bought the book, I assumed that I would read something like Fallen by Lauren Kate. I was wrong and I have not regretted it at all. Mainly character of the book is the 16-year-old Nora. A quiet girl, not in an annoying way, which for me was easy enough to understand her and her feelings. B... See full review
The Lost Symbol
USD$11.05 - Save $1.44 11% off - RRP $12.49
- Published: 22 Jul 2010
- Format: Paperback
Top review
The Lost Symbol!
written: 08 Sep 2010
The fact is that i wasn't planning to buy this book at all and i have never read before a book by Dan Brown (i have watched the movies for The Da Vinci Code and Angels&Demons though). When i got the book as present i thought "why not?". So here we are. The also known from the previous books pr... See full review
City of Bones
USD$11.56 - Save $1.43 11% off - RRP $12.99
- Published: 01 Mar 2008
- Format: Paperback
Review For the whole series!
written: 08 Sep 2010
Since i'm impatiently waiting for Clockwork Angel to arrive (finally was dispatched yesterday) i decided to do a sum-up review at Cassandra's Clare The Mortal Instruments series (sequel of the Infernal Devices which include Clockwork Angel). I will try not to spoil more than the summaries have alrea... See full review
Old Magic
USD$8.91 - Save $1.08 10% off - RRP $9.99
- Published: 22 Dec 2009
- Format: Paperback
Old Magic Review
written: 08 Sep 2010
This is one of the books that you remember for many reasons. For me, was the first book I've read about magic. One of the first I've read secretly, my parents had no clue since it was school season, while I was on the class-yes, during the lessons- and on the breaks. The first I've shared with my hi... See full review
The Named
USD$8.66 - Save $2.26 20% off - RRP $10.92
- Published: 03 May 2010
- Format: Paperback
Full Series Review
written: 08 Sep 2010
The main story is set in present years though some parts are in the past. We follow one by one the people that will become Guardians of Time starting with Ethan. Ethan has a long family history that we see to appear a lot in the book and we even get some glimpses of him before he get his power. Late... See full review
USD$6.99 - Save $3.93 35% off - RRP $10.92
- Published: 01 Apr 2007
- Format: Paperback
written: 08 Sep 2010
The Vampirates Series are set in the future at the year 2505. At that time there is a new era of piracy which is rising. Whoever control the seas, control an the world the say. We follow up the 16 year old twins, Connor and Grace and their life after their father died. While they try to escape from ... See full review
Clockwork Angel
- Published: 31 Aug 2010
- Format: Hardback
Clockwork Angel
written: 11 Sep 2010
I literally just finished the book. I wanted to write the review when is still fresh in my mind. To start with the environment i felt in love with it. It's one of the best settings i have read. When i was reading i was in London at 1878. I've never been there but the whole city, the atmosphere wa... See full review
written: 15 Sep 2010
This book was my last choice. I had plenty of money to spend (it was my birthday) and I was hoping from the title that is going to be a book for a guy who tries to figure out his sexuality. But I was wrong. Not fully, but still wrong. The book says about this high-school athlete who's shy and so ... See full review
- Sexy
Gentlemen and Players
USD$12.88 - Save $3.11 19% off - RRP $15.99
- Published: 01 Jan 2007
- Format: Paperback
written: 15 Sep 2010
I don't know exactly why I bought this book. It looked nice to have a book on my shelves that was normal, with simple people, no magicians, no genius children and so. Well, I have to admit that most of the books I thought they are simple, they are not. And this is an example. The book is telling ... See full review
The Burning Bridge
USD$8.64 - Save $0.72 (7%) - RRP $9.36
- Published: 02 Aug 2007
- Format: Paperback
written: 15 Sep 2010
The second book of the Ranger's Apprentice series takes us through the journeys of young Will throughout the lands of the Celts. This time seeking the aid of the Celt people against the evil Morgarath etc etc. There they will find a land torn and split as...well i shouldn't actually say due to spoil... See full review


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New lamps for Circus collection, by Mambo
Mambo Unlimited Ideas and designer Cláudia Melo presents Circus lamp collection: new versions in brass and lacquered white. An exquisite new version for Circus majestic lamps, just for ETTERO.
Besides its beautiful form, its light is very comfortable, because it has several abatjours and it becomes diffuse.
It turns out to be a piece that fulfill a whole room and make it ideal and comfy.

subset: commoncrawl


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Click Here to Watch the Whole Video in HD at Broke Straight Boys
These boys are very hot and very straight but most of all they are broke. Rather then lose their apartment, girlfriend, etc. they do sexual things with other guys for some quick cash.
Ashton is fucked by both Diesal and Mike R. Watch Ashton ride Mike like a rodeo champ while swallowing down Gigantor!
Pixels: 320X240 | Bitrate: 350K
Pixels: 640X480 | Bitrate: 700K
I lov how their comparing saggyness of nuts lmao
They need to release a scen where they all fuck


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It all started with Elliott Levy, executive director of the Aiken County Historical Museum.
At one of the Henry Middleton Chapter of Daughters of the American Revolution meetings, Levy had a piece of paper with the address of a young officer in Afghanistan who had volunteered time and service to the museum.
Elliott wanted to know if the chapter could help out with sending useful items from home. The note was passed on to Regent Judy Justice, who presented it at that meeting.
Every meeting is started with a ritual reciting that states: "To perpetuate the memory and spirit of men and women who achieved American independence, and to promote the development of an enlightened public opinion and to foster patriotic citizenship … these are the objects of our society, Daughters of the American Revolution."
This was a perfect project for the Henry Middleton Chapter to take on, and D.A.R. Service for Veterans Committee Chairman Candy Moore and Vice Chair Brenda McCabe got the ball rolling.
At the last summer luncheon meeting prior to break, chapter members met at The Wilcox Inn to swear in new chaplain, Marian McCrosson, and to bring goodies to send to Capt. Benjamin J. Van Meter. Women that didn't have items gave money for mailing.
The counter was piled high with bags of crew socks, snacks, single Crystal Light packets, current issues of magazines of all kinds men and women's alike. Also included werebooks on word search and Sudoku, T-shirts, preserves and chocolates.
In June, Moore received a letter of thanks. One of the paragraphs says it all, and it read as follows:
"I just wanted to let you all know that we have a couple of individuals on my team that are from South Carolina. Sergeant First Class William A. Brisbane is from James Island and Sergeant First Class Caprest C. Rhone is from Walterboro. Both are good men and have been very excited by the things that the good folks of Aiken have been sending us. In fact, one of our commanding general is from South Carolina as well. His name is Major General James M. Richardson, and he is originally from Myrtle Beach. Finally, we also have a unit here from the South Carolina Army National Guard. All said, there are plenty of "Carolina Boys" around here doing great things.
"We especially appreciate anything that reminds us of home. In one of the boxes sent to me, it contained a South Carolina flag, handmade potters, T-shirts, preserves and chocolates. All my Soldiers and I were very excited by all of those items. So, if it represents, is made in, or reminds us of South Carolina, then we will most certainly enjoy it. Again, thank you so very much for keeping us in your thoughts."
Benjamin J. Van Meter, Captain, U.S. Army
Van Meter, who has since been promoted to major and is now stationed in Baghdad, has been visiting Aiken since last week. The chapter is hosting a reception for him today at noon at the Aiken County Historical Museum.
Carolyn Hobbs Miller is registrar for the Henry Middleton Chapter of Daughters of the American Revolution in Aiken.
Photo submitted by Carolyn Miller The sideboard at The Willcox Inn is piled high with items headed for Afghanistan.×
Van Meter×
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Gear Question
How do they work with pads? I know some...
How do they work with pads? I know some have said that the Ex Officio underwear don't allow pads to stick to them. How do these work?
From my experience these work just fine with pads. ;)
View More Details on Patagonia Barely Bikini Underwear - Women's


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Come help us cheer on the UNH
Wildcats to victory!
The UNH Alumni Association and Athletics Department cordially invite you to join fellow alumni, parents and Wildcat fans in Albany (N.Y.) when the 12th-ranked University of New Hampshire men's hockey team competes in its ninth straight NCAA tournament! The third-seed Wildcats will square off against the second-seeded and sixth-ranked Cornell University Big Red on Friday, March 26 at 6:30 p.m. at the Times Union Center. (The winner advances to the regional final game on Saturday, March 27th against the winner of the Denver v. RIT game.)
We'll kick off Friday night at the Times Union Center with a pre-game reception from 5:00 to 6:30 p.m. – look for the "UNH Reception" signs on the second floor of the front Atrium. The Beast of the East UNH Pep Band will be performing at 5:30 along with an appearance of our very own mascot, Wild E Cat! UNH men's hockey coach Dick Umile '(72) will also stop by to talk to the crowd before the game! Come enjoy complimentary light refreshments and a cash bar. The game is only a few steps away inside the arena!
The Times Union Center is located on Pearl Street in downtown Albany, NY. For directions visit: http://timesunioncenter-albany.com/info/directions.aspx
• To purchase tickets, call the Times Union Center, Albany Ticket Sales Office at 1-518-487-2281.
• Ask for Maggie or Kenney to purchase "UNH Section" tickets.
• All tickets are RESERVED SEATS; there are no general admission tickets.
• There is a UNH block of hotel rooms at the Holiday Inn, Wolf Road in Albany, N.Y. at the special rate of $99.00 per night beginning Wednesday, March 24th through - Sunday, March 28th.
• You can reserve a hotel room by calling 1-800HOLIDAY and referencing the UNH Hockey Fan Block D2H
• This room block will be available until Friday, March 26, 2010, book early if you can.
• This is the same hotel that the UNH men's hockey team is staying at as well.
• Parking is available at Times Union Center garage, located behind the Center. There are also other garages in the immediate vicinity.
If you can't make it to the UNH/ Cornell NCAA tournament in Albany, NY, it will be aired live at 6:30 p.m. on ESPNU.
Come help us cheer on the UNH
Wildcats to victory!


text: To all and sundry, Searching through the archives didn't turnup any info. with regards to suggestions for OBSD artwork. So, if you'll indulge me, I'd like to suggest one here. My suggestion is somewhat along the lines of the Eddie 'The Trooper' poster from Iron Maiden. For those of you too young (or not into that type of music) to know what I'm talking about, here's a site with the poster: http://www.eddiesmegastore.com/ Then click on 'Posters', then 'The Trooper'. Now, Eddie is usually depicted as being angry, so the OBSD blowfish would have to tone that down. And the blood-dripping sword would also be a bit much, but the general context could be imported (in a more general and cartoonish way) for the OBSD artwork. I also was thinking that the Iron Maiden 'Aces High' poster would be a good OBSD scene (I don't have a link to show that one), but that's already kinda been done with the 2.7 artwork (only in a first person context). Just my two-cent suggestion. David __________________________________ Do you Yahoo!? Yahoo! SiteBuilder - Free, easy-to-use web site design software http://sitebuilder.yahoo.com


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July 29, 2015
In a St. Joseph Health Scene report, there's more evidence that sleeping too little or even too much may be bad for your heart. Researchers studied 58,000 men and women without heart disease. Those who slept less than 5 hours... or more than 9 hours.... were much more likely to die from cardiovascular disease over the next few years, compared to those who logged 7 hours a night.
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LOS ANGELES — They were the swinging, sassy voice of the home front for U.S. service personnel overseas during World War II, singing catchy hit tunes such as Boogie Woogie Bugle Boy that delighted Americans and catapulted the Andrews Sisters to the top of the pop charts.
One of the most-successful female recording groups in pop history, the sisters — LaVerne, Maxene and Patty Andrews — became a beloved American institution.
Yesterday, Patty Andrews, the group's lead singer and its last surviving member, died of natural causes at her home in the San Fernando Valley city of Northridge, according to her attorney. She was 94. Maxene, the middle Andrews sister, died in 1995 and LaVerne, the eldest, in 1967.
The Andrews Sisters began singing professionally in 1932. Known for their three-part harmonies and onstage humor, they churned out hit after hit: From 1938 to 1951, they had 19 gold records, dozens of top 10 singles and record sales of nearly 100 million.
Although Patty and Maxene lived near each other in California, according to reports they spoke rarely and saw each other just twice from 1974 to Maxene's death in 1995. Once was in 1987, when their star was dedicated on the Hollywood Walk of Fame.
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Outdoors Calendar: 03.23.08
In memory: GORDON POP SPEARS
The Coastal Bend outdoors community lost a longtime friend this past week. Gordon Pop Spears of Aransas Pass died March 18 at the age of 80. Pop was a gentleman and a friend to nearly everyone he met. Most duck hunters around here and beyond knew this graduate of Texas A&M University as the jovial patriarch of the Spears family hunting operations. Pop swore he began guiding when he was 7. His smile and his barbecue skills will be missed at the South Bay Outfitters hunting lodge.
The public is invited to the next meeting of the Rockport Fly Fishers March 27 at the Rockport Senior Center, 912 South Church St. The program begins at 6 p.m. with casting and tying demonstrations followed by a brief general meeting. Port O'Connor fly guide Jack Campbell is scheduled to give a presentation at 7:30 p.m. followed by a raffle and auction. The Rockport Fly Fishers also invite the public on first and third Wednesdays of each month for a free evening of fly tying, fly swapping and story telling at the Rockport Senior Center. Bring your own fly-tying stuff or just come to watch. Experienced fly tiers will be on hand. There will be a special table for beginner fly-tiers. Contact Ron Head at 386-0110 or firstname.lastname@example.com.
The Surfrider Foundation Texas Coastal Bend Chapter has a meeting scheduled for 6 p.m. March 31 in Room 1003 of the Carlos Truan Natural Resources Center at Texas A&M; University-Corpus Christi. At 7 p.m., Ben Rutledge is scheduled to speak on the proposed Mustang Island Reef Project, to discuss the possibility of creating an artificial reef here for surfing, fishing and scuba diving. Call 825-5759 or e-mail email@example.com.
Brighton Park Baptist Church, 3418 Airline Road, will host an outdoors expo from 10 a.m. to 4 p.m. April 5 at the corner of Airline Road and Brooke Road. This free event will feature fishing & hunting trophy showcases, a boat show, classic cars, motorcycles, fishing, retriever and hunting exhibits and demonstrations. A $10 men's steak & shrimp dinner will follow from 6-9 p.m., with guest speaker Jay Lowder and door prizes. The women's events are scheduled in the morning and evening. The $10 women's dinner from 6-9 p.m. features guest speaker Laurie Cole. Teen activities are scheduled from 5-8:30 p.m., featuring 3 on 3 basketball, a fashion show, extreme games, a mechanical bull and live performances by Christine Evans, They and H.D.W. Lil' Xhris. For younger children, all-day events include The Tumblebus, Hank Hough's Amazing Bird Dog Show, Rayz players, street hockey and more. Call 991-2910 or e-mail email@example.com.
The U.S Coast Guard Auxiliary, Flotilla 7-11, will host a basic boating course (ABS) from 7:45 a.m. to 5 p.m. April 19 at Seashore Middle Academy, 15437 SPID on Padre Island. This course is for adults and students 13-18. This course satisfies the state's certification requirement for boaters age 13-18. It'll cover safe boat handling and operations, basic seamanship, rules of the water, navigational aids, boating regulations, adverse conditions, VHF radio and trailering. The course fee is $45, which include the appropriate certificates. Registration is required and seating is limited. Call Brent Rourk at 244-7603. This class will be repeated in early June.
Sponsors and donations are needed for a charity fishing tournament sponsored by Catch the Cure to benefit Parkinson's research. The contest is scheduled for May 3 at Marker 37 Marina under the JFK Causeway. Organizers are looking for volunteers, auction items, cash, angler teams and sponsors. Entry fees are $185 for a 3-angler boat entry; $65 for no-boat individual angler; and $25 for junior anglers younger than 15. Entry includes a meal. Prizes will be awarded for heaviest trout, slot redfish, flounder, and heaviest other species, most redfish spots for juniors and more. Visit www.catchthecure.org or call Crystal at 726-1670 or Mona at 960-9597.
Everything SARGASSUM SYMPOSIUM
Come to the Sargassum Symposium May 12-14 at the Holiday Inn on North Padre Island. Topics will include weed removal, beach management, sagassum uses, recreational & economic implications and more.Info, visit www.sargassum.org or e-mail firstname.lastname@example.com.
Registration for Texas Brigade camps is under way. They're looking for adults volunteers and teen campers (13-17) interested in the outdoors, wildlife and conservation. Camps offer five-day programs. The fee is $300, but sponsorships are available. Each camp focuses on a specific program. Prospective adult volunteers need no experience. Call Helen at (800) 839-9453 or visit www.texasbrigades.org.
Subscribe to the Caller-Times
Need Help? Call us at 1-800-566-8098.
Monday-Friday: 6am-3pm / Saturday: 7am-11am / Sunday: 7am-11am


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ANCIENT OLYMPIA, Greece (September 29, 2013)--Using the sun's rays, the Olympic flame lighting was carried out without a hitch Sunday in southern Greece, ahead of the flame's journey across Russia's nine time zones and even a trip to space before the Feb. 7-23 Winter Games in Sochi.
The ceremony held Sunday included actresses dressed as ancient priestesses at the birthplace of the Greek games held in antiquity.
The flame was lit using a parabolic mirror.
NHL star Alex Ovechkin will be the first Russian involved in the torch relay.
Newly elected International Olympic Committee President Thomas Bach attended the ceremony Sunday.


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Step 1: Supplies!
the supplies you will need are:
~ soda boxes (approx. 1.5 per folder)
~ cutting utensil
~ packaging tape (clear)
~10-15 min.
Step 2: Find lateral surface of box
First, you will take your first box and cut through the middle to have a flattened box. Then cut off the edges. Also include the dotted line that you punchout if it is the fridge pack. Don't throw them away when your done though, you might use them for later.


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May 7, 2012
Contact: Kevin Coburn, Director of Communications
Green Mountain College Hosts 175th Commencement
POULTNEY--Green Mountain College's 2012 commencement speaker has been described as a powerful advocate for environmental justice and "the Rosa Parks of the green jobs movement." She is president of Majora Carter Group Consulting, and will address GMC graduates at the College's 175th commencement ceremony on Saturday, May 12 at 10 a.m. Carter will also receive an honorary doctor of laws degree from the College. Graduates of GMC's sustainable MBA and masters of science in environmental studies programs will also receive degrees.
The youngest of ten children growing up in the South Bronx, Carter graduated from Bronx High School of Science, studied film at Wesleyan University, and received an MFA from New York University in 1997. While at NYU, she had to return to her family's home in Hunts Point for financial reasons, inadvertently launching her career as a community visionary and entrepreneur.
She began volunteering for The Point Community Development Corporation, a youth development and arts organization. She secured a $10,000 grant to provide seed money for river access restoration projects along the Bronx River on land dominated by blighted post-industrial landscape. Working with other community groups, corporate neighbors, and the New York City Parks and Recreation department, Carter leveraged the grant into over $3.27 million in funding to build Hunts Point Riverside Park.
Carter founded and led Sustainable South Bronx (SSBx) from 2001-2008. This non-profit organization is dedicated to economic and environmental development informed by community needs under the guiding principle: "You shouldn't have to leave your neighborhood to live in a better one." Since 2008 The Majora Carter Group has developed climate adaptation, urban agribusiness, and leadership development strategies for business, government, foundations, universities and economically under-performing communities. Her many honors include a MacArthur "genius" Fellowship. She also hosts the Peabody Award-winning public radio series "The Promised Land."
Also receiving an honorary degree is Cornelius "Con" Hogan of Plainfield, Vermont. Hogan has devoted his career to providing humane and innovative solutions to social problems. He served as the Vermont secretary of human services from 1991 to 1999 and as commissioner for the Department of Corrections from 1977 to 1979. He is also a member of the Green Mountain Care Board which is charged with oversight of health care policy in the state. Hogan will receive an honorary doctor of humane letters degree.
The student speaker is Elizabeth Cerezo, a double-major in psychology and sociology/anthropology. She recently received a National Science Foundation Graduate Research Fellowship.
The outdoor ceremony on the Griswold Library lawn is open to the public (in the case of inclement weather, ticketing is required for the indoor venue. The ceremony will also be broadcast live via the Northeast Sports Network.


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Skip to Content
Home > Wellness Resources > Classes and Events > Event Search Results Detail
View All Events
Free Safety Seat inspections
Registration is closed.
No Appointments needed or taken. Certified CPS technicians will be on hand to check your car seats. Inspection includes: proper installation and child fit along with education on current child passenger safety regulations.
These free inspections are sponsored by Safe Kids Douglas County.
Please have your child's safety seat already installed upon arrival, and it will be inspected for safe and correct installation. Click on link for upcoming dates and times of appointment-only sessions as well as occasional drive-up checklane events where no appointment is required.
Events at Dale Willey Automotive are "open" events, meaning they don't require an appointment. Technicians work with families in the order that they arrive. The facility is a multi-lane garage, average wait time about 10 minutes.
Connect Care at 785-749-5800 or send an email
Infant CPR training
At this location Dale Willey Automotive Beginning on October 4, 2014


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Note: Each estimate of price flexibility shows the percentage change of a food price in response to a 1-percent increase in a food quantity.Source: USDA, ERS.
Last updated: Tuesday, June 05, 2012
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Usablenet Assistive is a Usablenet product. Usablenet Assistive Main Page.


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Strong multi-ply wood construction with plush, eco-friendly interior.
This Electric Bass Case by Road Runner features strong multi-ply wood construction with Tolex covering. The valances are stitched through the case top and your bass will be well kept in its soft, cushioned interior.
The soft, plush interior protects your bass from scratches and nicks, plus Road Runner uses a special, eco-friendly, dye-free material, which eliminates the release of polluting bi-products into the environment.


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Amateur black girl got fucked in the truck's front seat near to the public road with passing by cars!
Sexy teen rides the car driver
Lucky car driver get rided by a sexy teen passenger and fucks her on the front seat.
Amateur car front seat sex
Sexy teen (18+) brunette fucked at her boyfriend's car front seat by a tattooed bald guy! She moans like crazy - must to hear it
Gearshift sex on the street
Gear shift sex videos are really rare to find! I can say that I have the biggest shifter sex collection Today I found another video of hot lever stick fucking. The girl in not very beautiful, I mean she's not beautiful at all, but the clip is must to see! She fucks the shifter in a car parked right on the street! Watch her riding the car shifter and rubbing her wet pussy.
Beauty teen get paid to suck and fuck in car
How to trick young sexy girl to give you a car blowjob and then to ride you dick on the front seat? Just take some money nd offer a girl from the street. Then park your car in a city park and let her do the action! Maybe it will work, maybe it will not but that is the Money Talks scenario. Those guys every time find very sexy and innocent looking young girls! They also have good collection of car sex videos.
Nice tattooed amateur car fuck
Nice amateur action with a blonde teen. She gives the guy a nice blowjob in his car and after that rides his dick. The guy's dick is a wrinkled, ugly and soft, but the girl have nice tattooed body.
Dirty cop takes blowjob from a blonde teen in a police car
The clip is fake, of course, the poor thing is that the guy even don't look like a cop, but the girl is really cute. She suck shy, but with passion
Driving with naked babe on the passenger's seat
That european babe is really shameless! She gets naked in the car when her two friends driving and filming her daytime in the busy street traffic!
Girl squirts in a car
Hot brunette fucking herself with a g-spot toy until squirts.
Squiting girl in a car
Wet blonde squirts pussy juices all over the car!
Hot brunette fucked in a car for few dollars
Teen brunette have sex in a car for some bucks.
Amateur girl fucks boy in a car
One of the hottest amateur sex videos in the car. All in that video - blowjob, dick riding, masturbating and backseat banging.
Amateur sucking and fucking in a car
Amateur girl riding driver's cock and after that suck him.
Italian amateur blowjob in a car
Amateur girl from Rimini, Italy sucking cock in a car.
Blowjob with a facial in a car
Amateur blonde sucking cock ends with a facial in a car.


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Incentive pay approved for top UNC fundraiser post
The UNC Board of Trustees today approved an incentive compensation plan for the next vice chancellor for development, the university's top fundraiser.
The vice chancellor could receive incentive pay of as much as 25 percent of his or her base pay for meeting certain goals set by the chancellor.
UNC has launched a search for a permanent vice chancellor for development to replace Matt Kupec, who resigned last year after racking up nearly $17,000 in questionable travel-related and other expenses.
Julia Sprunt Grumbles, a former corporate vice president of Turner Broadcasting in Atlanta, has served as interim vice chancellor for development since September 2012.
UNC officials see the incentive as a valuable tool in the highly competitive search for senior level fundraising talent.
UNC General Administration has set a salary maximum for the position at $395,874. That means the school's top fundraiser could earn an extra $98,968 for meeting certain goals set by the university's chancellor.
Officials said the vice chancellor's goals would be reviewed annually and could change from year-to-year, depending on the needs of the university.
The board also approved significant changes to the Chapel Hill Foundation, Inc.'s travel and expense policies and procedures.


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Jiu-jitsu UniversityPaperback
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- Publisher: Victory Belt Publishing
- Format: Paperback | 368 pages
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- Publication date: 17 November 2008
- Publication City/Country: Auberry
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In "Jiu-Jitsu University", six-time Brazilian Jiu-Jitsu World Champion and two-time ADCC Submission Wrestling World Champion Saulo Ribeiro shows you why he is the teacher of champions. Even the more basic techniques come alive as Ribeiro sheds new light on theories and strategies previously thought antiquated. With a system that has been tried and tested at the highest levels of competition, Ribeiro will sculpt your foundation belt by belt and propel you toward jiu-jitsu excellence."Jiu-Jitsu University" is not your run-of-the-mill technique book. It is a detailed training manual that will ultimately change the way jiu-jitsu is taught around the globe. This book is tailor-made to complete the knowledge of any student, whether they are just starting out or already at the black belt level. His unique methodology combines new techniques with common misconceptions in Jiu-Jitsu, building a strong foundation to ensure that every reader has a firm basis in both the fundamental techniques as well as their advanced applications.
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Saulo Ribeiro is a Brazilian Jiu-Jitsu champion from the rough and beautiful Amazon-Manaus, Brazil. Not one to live within categories, Saulo's achievements go far beyond Jiu-Jitsu. He has gained acclaim as a lawyer and judge, and is now head instructor at the world-famous University of Jiu-Jitsu based out of San Diego, California. Kevin Howell is a political science professor based out of Huntington Beach, California. He holds a brown belt in Judo and a purple belt in Brazilian Jiu-Jitsu.
Customer reviews
By Fred Harrington 16 Oct 2010
I've had this about a year now, and have referred to it numerous times. It's a great reference book for the basics. It is by no means an exhaustive BJJ technique library but places emphasis on getting all the fundamentals down.
If I could only have one resource for my Gi game then this would be it without a doubt. Highly recommended.


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Megan Callaghan. Renewed Hope for the Incarcerated Through College Education.
This is a continued discussion about the Bard Prison Initiative with Dr. Megan Callaghan, the Director of Operations for BPI and a classroom instructor. This piece aired on October 19, 2014.
Inspiring Educators: Donnell Hughes. Bard Prison Initiative. A Second Chance.
This is a story of redemption. This is a story of Donnell Hughes. His story will challenge your concept of forgiveness, the value of education, the power of optimism, and the dedicated educators who have never forgotten the promise to the forgotten. This piece aired on October 6, 2014.
BPI Featured on NPR's Marketplace:
In Prison and Getting an Education
In a classroom, about a dozen men are discussing "The Odyssey," talking about the part where Odysseus comes home after being away for so long. It's hard not to imagine that the story has an added poignancy for them, students in the Bard College program at the Woodbourne Correctional Facility, a prison in upstate New York. This piece aired on May 26, 2014.
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NPR Ed Interview with a Recent BPI Alumnus:
A Former Drug Dealer Gives a Great Defense of the Liberal Arts
In preparation for my visit to the 11th annual commencement ceremony of the Bard Prison Initiative, I sat down for a conversation with Donnell Hughes, an alumnus of the program. BPI, as it's called, gives inmates at six prisons around New York state the opportunity to study in person with professors not only from Bard College, but from MIT, Harvard, Columbia, Vassar and local community colleges. This piece aired on June 21, 2014.
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N.Y. Governor Says College For Inmates Will Pay Of For Taxpayers
America used to have a robust college education system for prison inmates. It was seen as a way to rehabilitate men and women behind bars by helping them go straight when they got out. Those taxpayer-funded college classes were defunded in the 1990s. But New York Gov. Andrew Cuomo would like to bring them back in the state, prompting a fierce new debate over higher education in state prisons.
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Studies show that ex-offenders who are educated can get jobs and more easily turn their lives around in a positive direction. We are taking a big picture look at what it's like for people coming home from prison. What are the biggest challenges? What makes them go back to their old ways? What are the actions of
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about every aspect of this challenge that affects so many (mostly) young men.
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BPI Distinguished Fellow Ellen Lagemann was interviewed about BPI by Daria Fisk on UpFront – WMUA, 91.1 fm, December 12, 2012.
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An interview with BPI Executive Director Max Kenner and BPI Alumnus Justice Walston. Lopate, Kenner, and Walston discuss the withdrawal of public support for higher education in prisons during the mid-1990s, the subsequent entry of Bard College into the New York State prison system, and the merits of higher
education in prison. The piece aired on November 4, 2008.
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Bard Conservatory Orchestra Performs At Local Prison
Lara Pellegrinelli interviews BPI Executive Director Max Kenner, a BPI student, and a Bard College student in her story about the Bard Conservatory Orchestra's performance in Eastern Correctional Facility, which is believed to be the first time a full orchestra has performed within an American prison.
This piece aired on December 4, 2009.
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Turning Orderly Lives into Chaos
Terry Gross interviews two-time National Book Award nominee Scott Spencer, who talks about his experience teaching fiction writing for the Bard Prison Initiative. This piece aired on September 15, 2010.
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Another Look at Henry Cowell
John Schaefer and Leon Botstein discuss American composer Henry Cowell, crime, punishment, and the arts, as Botstein reflects on the Bard Prison Initiative. This piece aired on January 29, 2010.
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Cuomo Seeks Private Funds for "Prison University" Plan
Governor Cuomo dropped his plan to provide state-funded post-secondary education for New York State inmates, after some legislators rallied against the
proposal throughout late February. Now, reports say he is trying to find private funding for privately funded non-profits that already bring college tuition to state prisons. This piece aired on April 11, 2014.
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Bard College, PO Box 5000, Annandale-on-Hudson, NY 12504-5000 | 845-758-7308 |


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Courses: Microbiology (BIOLB255001)
Spring 2013
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Invisible to the naked eye, microbes occupy every niche on the planet. This course will examine how microbes have become successful colonizers; review aspects of interactions between microbes, humans and the environment; and explore practical uses of microbes in industry, medicine and environmental management. The course will combine lecture, discussion of primary literature and student presentations. Three hours of lecture and three hours of laboratory per week. Prerequisites: BIOL 110 and BIOL 111 or permission of the instructor.
Div: IIL; Approach: Scientific Investigation (SI); Haverford: Natural Science (NA)
Enrollment Limit: 20; Post Bacc Spaces: 3; Enrollment Criteria:
Sophomore; Junior; Senior; Post Bacc; Major/Minor/Concentration; Lottery;
Fulfills: Class Nbr: 1059 Div: IIL; SI;
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The Italian restaurant in Times Square is a magnet for theater-goers and celebrities alike.
Pranna Restaurant has agreed to hire additional security after complaints.
Tommy Lee Jones, Mimi Rogers and Dr. Ruth attended the screening at Chelsea's SVA Theatre.
The CFDA and "Made in NY" awards brought out Ben Stiller, Victor Cruz, Jessica Chastain and other celebs.
New York was largely overlooked at the Academy Awards this year.
A new exhibit at the Museum of Jewish Heritage traces the life of Emma Lazarus, who wrote "The New Colossus."
The stars came out for TriBeCa Film Festival screenings of "The Killer Inside Me" and "Please Give," as well as the Broadway opening of a musical about the Enron scandal.
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text:
The invention relates to novel magnetic resonance imaging contrast agents.
A—B is a structure selected from the group consisting of —CR2—CR2—, —CR═CR—, —CR2—CR2—CR2—, CR═CR—CR2— and —CR2—CR═CR, and wherein at least one A—B structure is —CR═CR—;
X1 and X2 are independently selected from the group consisting of CR2COOH, and CR(CR2COOH)2;
each R is independently selected from the group consisting of hydrogen, alkyl, aryl, alcohol, amine, amido, nitro, ether, ester, ketone, imino, aldehyde, alkoxy, carbonyl, halogen, sulfur containing moiety, phosphorus containing moiety, targeting moiety, or, together with an adjacent R group forms an alkyl or aryl group; or a salt thereof.
A—B is a structure selected from the group consisting of —CR2—CR2—, —CR═CR—, —CR2—CR2—CR2—, CR═CR—CR2— and —CR2—CR═CR, and wherein at least one A—B structure is —CR═CR—;
X1 and X2 are independently selected from the group consisting of CR2 COO−, and CR(CR2COO−)2;
each R is independently selected from the group consisting of hydrogen, alkyl, aryl, alcohol, amine, amido, nitro, ether, ester, ketone, imino, aldehyde, alkoxy, carbonyl, halogen, sulfur containing moiety, phosphorus containing moiety, targeting moiety, or, together with an adjacent R group forms an alkyl or aryl group; and a paramagnetic ion; or a salt thereof.
3. An MRI composition according to
5. A method of magnetic resonance imaging of a cell, tissue or patient comprising administering an MRI agent according to
6. An MRI composition according to
7. An MRI composition according to
8. An MRI composition according to
9. An MRI composition according to
10. An MRI composition according to
This is a continuing application of U.S. Ser. No. 60/218,581, filed Jul. 17, 2000.
The U.S. government has certain rights in this invention pursuant to Grant No. RO 1AI47003 awarded by the National Institute of Health.
The invention relates to novel magnetic resonance imaging contrast agents.
Magnetic resonance imaging (MRI) is a diagnostic and research procedure that uses high magnetic fields and radio-frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical "spin" of the water proton nuclei that ultimately gives rise to the signal in all imaging experiments. In MRI the sample to be imaged is placed in a strong static magnetic field (1-12 Tesla) and the spins are excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. MRI is able to generate structural information in three dimensions in relatively short time spans.
The Image.
MR images are typically displayed on a gray scale with black the lowest and white the highest measured intensity (I). This measured intensity I=C*M, where C is the concentration of spins (in this case, water concentration) and M is a measure of the magnetization present at time of the measurement. Although variations in water concentration (C) can give rise to contrast in MR images, it is the strong dependence of the rate of change of M on local environment that is the source of image intensity variation in MRI. Two characteristic relaxation times, T1 & T2, govern the rate at which the magnetization can be accurately measured. T1 is the exponential time constant for the spins to decay back to equilibrium after being perturbed by the RF pulse. In order to increase the signal-to-noise ratio (SNR) a typical MR imaging scan (RF & gradient pulse sequence and data acquisition) is repeated at a constant rate for a predetermined number of times and the data averaged. The signal amplitude recorded for any given scan is proportional to the number of spins that have decayed back to equilibrium since the previous scan. Thus, regions with rapidly decaying spins (i.e. short T1 values) will recover all of their signal amplitude between successive scans.
The measured intensities in the final image will accurately reflect the spin density (i.e. water content). Regions with long T1 values compared to the time between scans will progressively lose signal until a steady state condition is reached and will appear as darker regions in the final image. Changes in T2 (spin-spin relaxation time) result in changes in the signal linewidth (shorter T2 values) yielding larger linewidths. In extreme situations the linewidth can be so large that the signal is indistinguishable from background noise. In clinical imaging, water relaxation characteristics vary from tissue to tissue, providing the contrast which allows the discrimination of tissue types. Moreover, the MRI experiment can be setup so that regions of the sample with short T1 values and/or long T2 values are preferentially enhanced so called T1-weighted and T2-weighted imaging protocol.
MRI Contrast Agents.
There is a rapidly growing body of literature demonstrating the clinical effectiveness of paramagnetic contrast agents (currently 8 are in clinical trials or in use). The capacity to differentiate regions/tissues that may be magnetically similar but histologically distinct is a major impetus for the preparation of these agents [1, 2]. In the design of MRI agents, strict attention must be given to a variety of properties that will ultimately effect the physiological outcome apart from the ability to provide contrast enhancement [3]. Two fundamental properties that must be considered are biocompatability and proton relaxation enhancement. Biocompatability is influenced by several factors including toxicity, stability (thermodynamic and kinetic), pharmacokinetics and biodistribution. Proton relaxation enhancement (or relaxivity) is chiefly governed by the choice of metal and rotational correlation times.
The first feature to be considered during the design stage is the selection of the metal atom, which will dominate the measured relaxivity of the complex. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent relaxation enhancement agents. They decrease the T1 and T2 relaxation times of nearby (r6 dependence) spins. Some paramagnetic ions decrease the T1 without causing substantial linebroadening (e.g. gadolinium (III), (Gd3+)), while others induce drastic linebroadening (e.g. superparamagnetic iron oxide). The mechanism of T1 relaxation is generally a through space dipole-dipole interaction between the unpaired electrons of the paramagnet (the metal atom with an unpaired electron) and bulk water molecules (water molecules that are not "bound" to the metal atom) that are in fast exchange with water molecules in the metal's inner coordination sphere (are bound to the metal atom).
For example, regions associated with a Gd3+ ion (near-by water molecules) appear bright in an MR image where the normal aqueous solution appears as dark background if the time between successive scans in the experiment is short (i.e. T1 weighted image). Localized T2 shortening caused by superparamagnetic particles is believed to be due to the local magnetic field inhomogeneities associated with the large magnetic moments of these particles. Regions associated with a superparamagnetic iron oxide particle appear dark in an MR image where the normal aqueous solution appears as high intensity background if the echo time (TE) in the spin-echo pulse sequence experiment is long (i.e. T2-weighted image). The lanthanide atom Gd3+ is by the far the most frequently chosen metal atom for MRI contrast agents because it has a very high magnetic moment (u2=63BM2), and a symmetric electronic ground state, (S8). Transition metals such as high spin Mn(II) and Fe(III) are also candidates due to their high magnetic moments.
Once the appropriate metal has been selected, a suitable ligand or chelate must be found to render the complex nontoxic. The term chelator is derived from the Greek word chele which means a "crabs claw", an appropriate description for a material that uses its many "arms" to grab and hold on to a metal atom (see DTPA below). Several factors influence the stability of chelate complexes include enthalpy and entropy effects (e.g. number, charge and basicity of coordinating groups, ligand field and conformational effects). Various molecular design features of the ligand can be directly correlated with physiological results. For example, the presence of a single methyl group on a given ligand structure can have a pronounced effect on clearance rate. While the addition of a bromine group can force a given complex from a purely extracellular role to an effective agent that collects in hepatocytes.
Diethylenetriaminepentaacetic (DTPA) chelates and thus acts to detoxify lanthanide ions. The stability constant (K) for Gd(DTPA)2− is very high (logK=22.4) and is more commonly known as the formation constant (the higher the logK, the more stable the complex). This thermodynamic parameter indicates the fraction of Gd3+ ions that are in the unbound state will be quite small and should not be confused with the rate (kinetic stability) at which the loss of metal occurs (kf/kd). The water soluble Gd(DTPA)2− chelate is stable, nontoxic, and one of the most widely used contrast enhancement agents in experimental and clinical imaging research. It was approved for clinical use in adult patients in June of 1988. It is an extracellular agent that accumulates in tissue by perfusion dominated processes.
To date, a number of chelators have been used, including diethylenetriaminepentaacetic (DTPA), 1,4,7,10-tetraazacyclododecane′-N,N′N″,N′″-tetracetic acid (DOTA), and derivatives thereof. See U.S. Pat. Nos. 5,155,215, 5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532, and Meyer et al., Invest. Radiol. 25: S53 (1990).
Image enhancement improvements using Gd(DTPA) are well documented in a number of applications (Runge et al., Magn, Reson. Imag. 3:85 (1991); Russell et al., AJR 152:813 (1989); Meyer et al., Invest. Radiol. 25:S53 (1990)) including visualizing blood-brain barrier disruptions caused by space occupying lesions and detection of abnormal vascularity. It has recently been applied to the functional mapping of the human visual cortex by defining regional cerebral hemodynamics (Belliveau 35 et al., (1991) 254:719).
Another chelator used in Gd contrast agents is the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-N,N′,N″N′″-tetracetic acid (DOTA). The Gd-DOTA complex has been thoroughly studied in laboratory tests involving animals and humans. The complex is conformationally rigid, has an extremely high formation constant (logk=28.5), and at physiological pH possess very slow dissociation kinetics. Recently, the GdDOTA complex was approved as an MRI contrast agent for use in adults and infants in France and has been administered to over 4500 patients.
Accordingly, it is an object of the invention to provide further examples of DOTA derivatives for use in MRI imaging.
In accordance with the objects outlined above, the present invention provides chelates having the formula:
A—B is a structure selected from the group consisting of —CR2—CR2—, —CR═CR—, —CR2—CR2—CR2—, CR═CR—CR2— and —CR2—CR═CR—;
X1 and X2 are independently selected from the group consisting of CR2 COO−, CR 2COOH, CR(CR2COO−)2, CR(CR2COOH)2;
each R is independently selected from the group consisting of hydrogen, alkyl, aryl, alcohol, amine, amido, nitro, ether, ester, ketone, imino, aldehyde, alkoxy, carbonyl, halogen, sulfur containing moiety, phosphorus containing moiety, targeting moiety, moiety, or, together with an adjacent R group forms an alkyl or aryl group.
In an additional aspect, the invention provides chelates having the formula:
wherein each n is an integer independently selected from one to three.
In a further aspect, the invention provides compositions comprising a chelate of the invention complexed with a paramagnetic ion such as gadolinium, manganese or iron.
In an additional aspect, the invention provides compositions having the formula:
In a further aspect, the invention provides compositions having the formula:
each n is an integer independently selected from 1 to 3.
In an additional aspect, the invention provides chelates having the formula:
In a further aspect, the invention provides methods of magnetic resonance imaging of a cell, tissue or patient comprising administering an MRI agent of the invention to a cell, tissue or patient and rendering a magnetic resonance image of said cell, tissue or patient.
FIGS. 1A-1E depict a variety of preferred embodiments utilizing macrocycles with four coordination atoms in the macrocycle. FIG. 1A depicts Structure 1 of the invention. FIGS. 1B-1E depict a variety of chelators of the invention, without the metal ions.
FIGS. 2A-2E depict a variety of suitable —A—B— groups, any one of which can be included in the macrocycles of the invention.
FIG. 3 depicts the synthetic schemes of the invention.
The present invention provides novel magnetic resonance imaging contrast agents based on the DOTA structure, which can provide better and more stable MRI contrast agents. Cross-bridged tetraazamacrocycles have been shown to form complexes with transition metal ions having unprecedented kinetic stability, even though they do not fully saturate the metal's coordination sites. See Hubin et al., J. Chem. Soc. Chem, Commun, 1998:1675; Hubin et al., Inorg. Chem, 1999, 38:4435; Hubin et al., J. Am. Chem. Soc. 2000,122:2512; WO 98/39098 and WO 98/39046, all of which are expressly incorporated by reference.
The complexes of the invention comprise a chelator and a paramagnetic metal ion bound to the chelator. By "paramagnetic metal ion", "paramagnetic ion" or "metal ion" herein is meant a metal ion which is magnetized parallel or antiparallel to a magnetic field to an extent proportional to the field.
Generally, these are metal ions which have unpaired electrons; this is a term understood in the art. Examples of suitable paramagnetic metal ions, include, but are not limited to, gadolinium III (Gd+3 or Gd(III)), iron III (Fe+3 or Fe(III)), manganese II (Mn+2 or Mn(II)), yttrium III (Yt+3 or Yt(III)), dysprosium (Dy+3 or Dy(III)), and chromium (Cr(III) or Cr+3). In a preferred embodiment the paramagnetic ion is the lanthanide atom Gd(III), due to its high magnetic moment (u2=63BM2), a symmetric electronic ground state (S8), and its current approval for diagnostic use in humans.
In addition to the metal ion, the metal ion complexes of the invention comprise a chelator. Due to the relatively high toxicity of many of the paramagnetic ions, the ions are rendered nontoxic in physiological systems by binding to a suitable chelator as outlined herein.
Accordingly, the present invention provides a number of suitable chelators for use in the present invention. The chelators shown below do not depict the chelated metal ion, although as will be appreciated by those in the art, they may be present as well.
In a preferred embodiment, the chelators have the structure depicted below:
In some embodiments, the nitrogen atoms of the ring may be independently replaced with either oxygen or sulfur, depending on the metal ion; however, when the preferred Gd+3 is used, nitrogen atoms are preferred.
In a preferred embodiment, the —A—B— moiety is an alkyl moiety of C1-5, with alkyl, alkene, and alkyne bonds possible. In preferred embodiments, the —A—B— moiety is an alkyl of C2-3 selected from the group consisting of —CR2—CR2—, —CR═CR—, —CR2—CR2—CR2—, —CR═CR—CR2— and —CR2—CR═CR— as is depicted in FIG. 2. The R substitution groups are as defined below.
The X groups of the invention (sometimes referred to herein as the "arms" of the chelator) generally provide one or more additional coordination atoms. Again, the choice of the coordination atom will depend on the metal ion used, with —COOH groups being a coordination moiety of preference with Gd+3. In some embodiments, straight alkyl chains (including both substituted alkyl, heteroalkyl and substituted heteroalkyl) are used. In alternate embodiments, the X groups depicted herein can be branched alkyl structures, with a single alkyl group comprising at least one carbon atom branching to provide multiple coordination atoms as depicted in Structure 3, below.
In general, when not attached, the X groups of the Structures are independently selected from the group consisting of —(CR2)nCOO−, —(CR2)nCOOH, —CR(CR2COO−)2, —CR(CR2COOH)2, —(CR2)n—CR((CR2)m—COOH)2, —(CR2)n—CR((CR2)m—COO−)2, —(CR2)n—C(CR2)m—COOH)3; and —C((CR2)n—COOH)3. In this embodiment, n is an integer from 1 to 5, with 1, 2 and 3 being preferred; m is an integer from 0 to 5, with 0, 1 and 2 being preferred.
It is to be understood that the exact composition of the X groups will depend on the presence of the metal ion. That is, the hydrogen atoms of the coordination group can be present in the absence of the metal ion, but are absent in the presence of the metal ion. Thus, the structures depicted herein can be shown with the hydrogen atoms on the carboxyl group (in the absence of the metal ion) or without them (in the presence of the metal ion). Thus, preferred embodiments have the structure shown in Structure 2:
In this embodiment, n is an integer from one to five, with from one to three being preferred, and one and two being particularly preferred. Again, the carboxy group is shown without the hydrogen of the hydroxyl group, which will depend on the presence or absence of the metal ion.
A preferred embodiment with branching X groups is depicted in Structure 3, below:
Structure 3 is shown with particular —A—B— groups, all R groups as hydrogen, and no metal ion.
In a preferred embodiment at least one of the R groups attached to the "arms" of the chelator (e.g. the X groups) comprises an alkyl (including substituted and heteroalkyl groups), or aryl (including substituted and heteroaryl groups), i.e. is a group sterically bulkier than hydrogen. Preferred groups include the C1 through C6 alkyl groups with methyl being particularly preferred.
However the inclusion of too many groups may drive the equilibrium in the other direction. Therefore in a preferred embodiment only 1 or 2 of these positions is a non-hydrogen group, unless other methods are used to drive the equilibrium towards binding.
In the figures and agents described herein, R is independently selected from the group consisting of hydrogen, alkyl, aryl, alcohol, amine, amido, nitro, ether, ester, ketone, imino, aldehyde, alkoxy, carbonyl, halogen, sulfur containing moiety, phosphorus containing moiety, targeting moiety, or, together with an adjacent R group forms an alkyl or aryl group.
As will be appreciated by those skilled in the art, many positions designated herein and in the structures of the figures may have more than 1 R group attached, depending on the valency of the atoms; generally, carbon atoms that are not participating in double bonds can have two R groups attached (R′ and R″), although in a preferred embodiment only a single non-hydrogen R group is attached at any particular position; that is, preferably at least one of the R groups at each position is hydrogen. Thus, if R is an alkyl or aryl group, there is generally an additional hydrogen attached to the carbon, although not depicted herein. In many preferred embodiments, all the R groups are hydrogen, with the exception of targeting and blocking moieties.
By "alkyl group" or grammatical equivalents herein is meant a straight or branched chain alkyl group, with straight chain alkyl groups being preferred. If branched, it may be branched at one or more positions, and unless specified, at any position. Also included within the definition of alkyl are heteroalkyl groups, wherein the heteroatom is selected from nitrogen, oxygen, phosphorus, sulfur and silicon. Also included within the definition of an alkyl group are cycloalkyl groups such as C5 and C6 rings, and heterocycloalkyl.
Additional suitable heterocyclic substituted rings are depicted in U.S. Pat. No. 5,087,440, expressly incorporated by reference. In some embodiments, two adjacent R groups may be bonded together to form ring structures together with the carbon atoms of the chelator, such as is described in U.S. Pat. No. 5,358,704, expressly incorporated by reference. These ring structures may be similarly substituted.
The alkyl group may range from about 1 to 20 carbon atoms (C1-C20), with a preferred embodiment utilizing from about 1 to about 10 carbon atoms (C1-C10), with about C1 through about C5 being preferred. However, in some embodiments, the alkyl group may be larger, for example when the alkyl group is the coordination site barrier.
By "alkyl amine" or grammatical equivalents herein is meant an alkyl group as defined above, substituted with an amine group at any position. In addition, the alkyl amine may have other substitution groups, as outlined above for alkyl group. The amine may be primary (—NH2R), secondary (—NHR2), or tertiary (—NR3). When the amine is a secondary or tertiary amine, suitable R groups are alkyl groups as defined above. A preferred alkyl amine is p-aminobenzyl. When the alkyl amine serves as the coordination site barrier, as described below, preferred embodiments utilize the nitrogen atom of the amine as a coordination atom, for example when the alkyl amine includes a pyridine or pyrrole ring.
By "aryl group" or grammatical equivalents herein is meant aromatic aryl rings such as phenyl, heterocyclic aromatic rings such as pyridine, furan, thiophene, pyrrole, indole and purine, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus.
Included within the definition of "alkyl" and "aryl" are substituted alkyl and aryl groups. That is, the alkyl and aryl groups may be substituted, with one or more substitution groups. For example, a phenyl group may be a substituted phenyl group. Suitable substitution groups include, but are not limited to, halogens such as chlorine, bromine and fluorine, amines, hydroxy groups, carboxylic acids, nitro groups, carbonyl and other alkyl and aryl groups as defined herein. Thus, arylalkyl and hydroxyalkyl groups are also suitable for use in the invention. Preferred substitution groups include alkyl amines and alkyl hydroxy.
In some embodiments, adjacent R groups can be joined to form cyclic structures, either cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, or pluricyclic structures comprising a combination of these ring structures, including substituted derivatives of any of these. Similarly, when the R groups are alkyl and aryl groups, pluricyclic groups including cycloalkyl, cycloheteroalkyl, aryl, heteroaryl and substituted derivatives can also be used.
By "amino groups" or grammatical equivalents herein is meant —NH2, —NHR and —NR2 groups, with R being as defined herein.
By "nitro group" herein is meant an —NO2 group.
By "sulfur containing moieties" herein is meant compounds containing sulfur atoms, including but not limited to, thia-, thio- and sulfo- compounds, thiols (—SH and —SR), and sulfides (—RSR—). By "phosphorus containing moieties" herein is meant compounds containing phosphorus, including, but not limited to, phosphines and phosphates. By "silicon containing moieties" herein is meant compounds containing silicon.
By "ether" herein is meant an —O—R group. Preferred ethers include alkoxy groups, with —O—(CH2)2CH3 and —O—(CH2)4CH3 being preferred.
By "ester" herein is meant a —COOR group.
By "halogen" herein is meant bromine, iodine, chlorine, or fluorine. Preferred substituted alkyls are partially or fully halogenated alkyls such as CF3, etc.
By "aldehyde" herein is meant —RCHO groups.
By "alcohol" or "alkoxy" herein is meant —OH groups, and alkyl alcohols —ROH.
By "amido" herein is meant —RCONH— or RCONR— groups.
By "ethylene glycol" or "(poly)ethylene glycol" herein is meant a —(O—CH2—CH2)n— group, although each carbon atom of the ethylene group may also be singly or doubly substituted, i.e. —(O—CR2—CR2)n—, with R as described above. Ethylene glycol derivatives with other heteroatoms in place of oxygen (i.e. —(N—CH2—CH2)n— or —(S—CH2—CH2)n—, or with substitution groups) are also preferred.
By "ketone" herein is meant —R—CO—R—.
By "imino group" herein is meant —C—NH—C.
By "carbonyl" herein is meant —C═O.
By "phosphorous moieties" herein is meant moieties containing the —PO(OH)(R)2 group. The phosphorus may be an alkyl phosphorus; for example, DOTEP utilizes ethylphosphorus as a substitution group on DOTA. R is as defined above, with preferred embodiments utilizing alkyl, substituted alkyl and hydroxy. A preferred embodiment has a —PO(OH)2R group.
In addition, the complexes and metal ion complexes of the invention may further comprise one or more targeting moieties; i.e. one or more R groups may be a targeting moiety. That is, a targeting moiety may be attached at any of the R positions (or to a linker, including a polymer, or to a blocking moiety, etc.), although in a preferred embodiment the targeting moiety does not replace a coordination atom. By "targeting moiety" herein is meant a functional group which serves to target or direct the complex to a particular location, cell type, diseased tissue, or association. In general, the targeting moiety is directed against a target molecule. As will be appreciated by those in the art, the MRI contrast agents of the invention are generally injected intraveneously; thus preferred targeting moieties are those that allow concentration of the agents in a particular localization. Thus, for example, antibodies, cell surface receptor ligands and hormones, lipids, sugars and dextrans, alcohols, bile acids, fatty acids, amino acids, peptides and nucleic acids may all be attached to localize or target the contrast agent to a particular site.
In a preferred embodiment, the targeting moiety allows targeting of the MRI agents of the invention to a particular tissue or the surface of a cell.
As will be appreciated by those in the art, the targeting moieties can be attached in a large number of different ways, and in a variety of configurations.
In a preferred embodiment, the targeting moiety is a peptide. For example, chemotactic peptides have been used to image tissue injury and inflammation, particularly by bacterial infection; see WO 97/14443, hereby expressly incorporated by reference in its entirety.
In a preferred embodiment, the targeting moiety is an antibody. The term "antibody" includes antibody fragments, as are known in the art, including Fab Fab2, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
In a preferred embodiment, the antibody targeting moieties of the invention are humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin 10 [Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage 25 display libraries [Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10:779-783 35 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a first target molecule and the other one is for a second target molecule.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
In a preferred embodiment, the antibody is directed against a cell-surface marker on a cancer cell; that is, the target molecule is a cell surface molecule. As is known in the art, there are a wide variety of antibodies known to be differentially expressed on tumor cells, including, but not limited to, HER2, VEGF, etc.
In addition, antibodies against physiologically relevant carbohydrates may be used, including, but not limited to, antibodies against markers for breast cancer (CA 15-3, CA 549, CA 27.29), mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA 125), pancreatic cancer (DE-PAN-2), and colorectal and pancreatic cancer (CA 19, CA 50, CA242).
In a preferred embodiment, the targeting moiety is all or a portion (e.g. a binding portion) of a ligand for a cell surface receptor. Suitable ligands include, but are not limited to, all or a functional portion of the ligands that bind to a cell surface receptor selected from the group consisting of insulin receptor (insulin), insulin-like growth factor receptor (including both IGF-1 and IGF-2), growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), transferrin receptor (transferrin), epidermal growth factor receptor (EGF), low density lipoprotein receptor, high density lipoprotein receptor, leptin receptor, estrogen receptor (estrogen); interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGF receptor (VEGF), PDGF receptor (PDGF), transforming growth factor receptor (including TGF-α and TGF-β), EPO receptor (EPO), TPO receptor (TPO), ciliary neurotrophic factor receptor, prolactin receptor, and T-cell receptors. In particular, hormone ligands are preferred. Hormones include both steroid hormones and proteinaceous hormones, including, but not limited to, epinephrine, thyroxine, oxytocin, insulin, thyroid-stimulating hormone, calcitonin, chorionic gonadotropin, cortictropin, follicle-stimulating hormone, glucagon, leuteinizing hormone, lipotropin, melanocyte-stimutating hormone, norepinephrine, parathryroid hormone, thyroid-stimulating hormone (TSH), vasopressin, enkephalins, seratonin, estradiol, progesterone, testosterone, cortisone, and glucocorticoids and the hormones listed above. Receptor ligands include ligands that bind to receptors such as cell surface receptors, which include hormones, lipids, proteins, glycoproteins, signal transducers, growth factors, cytokines, and others.
In a preferred embodiment, the targeting moiety is a carbohydrate. By "carbohydrate" herein is meant a compound with the general formula Cx(H2O)y. Monosaccharides, disaccharides, and oligo- or polysaccharides are all included within the definition and comprise polymers of various sugar molecules linked via glycosidic linkages. Particularly preferred carbohydrates are those that comprise all or part of the carbohydrate component of glycosylated proteins, including monomers and oligomers of galactose, mannose, fucose, galactosamine, (particularly N-acetylglucosamine), glucosamine, glucose and sialic acid, and in particular the glycosylation component that allows binding to certain receptors such as cell surface receptors. Other carbohydrates comprise monomers and polymers of glucose, ribose, lactose, raffinose, fructose, and other biologically significant carbohydrates. In particular, polysaccharides (including, but not limited to, arabinogalactan, gum arabic, mannan, etc.) have been used to deliver MRI agents into cells; see U.S. Pat. No. 5,554,386, hereby incorporated by reference in its entirety.
In a preferred embodiment, the targeting moiety is a lipid. "Lipid" as used herein includes fats, fatty oils, waxes, phospholipids, glycolipids, terpenes, fatty acids, and glycerides, particularly the triglycerides. Also included within the definition of lipids are the eicosanoids, steroids and sterols, some of which are also hormones, such as prostaglandins, opiates, and cholesterol.
In a preferred embodiment, the targeting moiety is selected from the group consisting of enzyme substrates or inhibitors, receptor ligands, antibodies, antigens, ion binding compounds, substantially complementary nucleic acids, nucleic acid binding proteins, etc.
In a preferred embodiment, the targeting moiety may be used to either allow the internalization of the MRI agent to the cell cytoplasm or localize it to a particular cellular compartment, such as the nucleus.
In a preferred embodiment, the targeting moiety is all or a portion of the HIV-1 Tat protein, and analogs and related proteins, which allows very high uptake into target cells. See for example, Fawell et al., PNAS USA 91:664 (1994); Frankel et al., Cell 55:1189 (1988); Savion et al., J. Biol. Chem. 256:1149 (1981); Derossi et al., J. Biol. Chem. 269:10444 (1994); and Baldin et al., EMBO J. 9:1511 (1990), all of which are incorporated by reference.
In a preferred embodiment, the targeting moiety is a nuclear localization signal (NLS). NLSs are generally short, positively charged (basic) domains that serve to direct the moiety to which they are attached to the cell's nucleus. Numerous NLS amino acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys Arg Lys Val), Kalderon (1984), et al., Cell, 39:499-509; the human retinoic acid receptor-β nuclear localization signal (ARRRRP); NFKB p50 (EEVQRKRQKL; Ghosh et al., Cell 62:1019 (1990); NFKB p65 (EEKRKRTYE; Nolan et al., Cell 64:961 (1991); and others (see for example Boulikas, J. Cell. Biochem. 55(1):32-58 (1994), hereby incorporated by reference) and double basic NLS's exemplified by that of the Xenopus (African clawed toad) protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly GIn Ala Lys Lys Lys Lys Leu Asp), Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall, et al., J. Cell Biol., 107:641-849; 1988). Numerous localization studies have demonstrated that NLSs incorporated in synthetic peptides or grafted onto reporter proteins not normally targeted to the cell nucleus cause these peptides and reporter proteins to be concentrated in the nucleus. See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol., 2:367-390, 1986; Bonnerot, et al., Proc. Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci. USA, 87:458-462, 1990.
In a preferred embodiment, targeting moieties for the hepatobiliary system are used; see U.S. Pat. Nos. 5,573,752 and 5,582,814, both of which are hereby incorporated by reference in their entirety.
In a preferred embodiment, the metal ion complexes of the present invention are water soluble or soluble in aqueous solution. By "soluble in aqueous solution" herein is meant that the MRI agent has appreciable solubility in aqueous solution and other physiological buffers and solutions. Solubility may be measured in a variety of ways. In one embodiment, solubility is measured using the United States Pharmacopeia solubility classifications, with the metal ion complex being either very soluble (requiring less than one part of solvent for 1 part of solute), freely soluble (requiring one to ten parts solvent per 1 part solute), soluble (requiring ten to thirty parts solvent per 1 part solute), sparingly soluble (requiring 30 to 100 parts solvent per 1 part solute), or slightly soluble (requiring 100-1000 parts solvent per 1 part solute).
Testing whether a particular metal ion complex is soluble in aqueous solution is routine, as will be appreciated by those in the art. For example, the parts of solvent required to solubilize a single part of MRI agent may be measured, or solubility in gm/ml may be determined.
In a preferred embodiment, the MRI contrast agents of the invention comprise more than one metal ion, such that the signal is increased. As is outlined below, this may be done in a number of ways, including, but not limited to, the use of multiple metal ions in a single chelate, the use of a single blocking moiety to block more than one chelated metal ion, or the oligomerization of the agents of the invention, including both multimers and the use of polymeric linkers to attach agents together.
In a preferred embodiment, the MRI agents of the invention comprise at least two paramagnetic metal ions, each with a chelator; that is, multimeric MRI agents are made. In a preferred embodiment, the chelators are linked together, either directly or through the use of a linker such as a coupling moiety or polymer. For example, using substitution groups that serve as functional groups for chemical attachment on the chelator, attachment to other chelators may be accomplished.
In a preferred embodiment, the chelators of the invention include one or more substitution groups that serve as functional groups for chemical attachment. Suitable functional groups include, but are not limited to, amines (preferably primary amines), carboxy groups, and thiols (including SPDP, alkyl and aryl halides, maleimides, α-haloacetyls, and pyridyl disulfides) are useful as functional groups that can allow attachment.
In one embodiment, the chelators are linked together directly, using at least one functional group on each chelator. This may be accomplished using any number of stable bifunctional groups well known in the art, including homobifunctional and heterobifunctional linkers (see Pierce Catalog and Handbook, 1994, pages T155-T200, hereby expressly incorporated by reference). This may result in direct linkage, for example when one chelator comprises a primary amine as a functional group and the second comprises a carboxy group as the functional group, and carbodiimide is used as an agent to activate the carboxy for attach by the nucleophilic amine (see Torchilin et al., Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991). Alternatively, as will be appreciated by those in the art, the use of some bifunctional linkers results in a short coupling moiety being present in the structure. A "coupling moiety" is capable of covalently linking two or more entities. In this embodiment, one end or part of the coupling moiety is attached to the first MRI contrast agent, and the other is attached to the second MRI agent. The functional group(s) of the coupling moiety are generally attached to additional atoms, such as alkyl or aryl groups (including hetero alkyl and aryl, and substituted derivatives), to form the coupling moiety. Oxo linkers are also preferred. As will be appreciated by those in the art, a wide range of coupling moieties are possible, and are generally only limited by the ability to synthesize the molecule and the reactivity of the functional group. Generally, the coupling moiety comprises at least one carbon atom, due to synthetic requirements; however, in some embodiments, the coupling moiety may comprise just the functional group.
In a preferred embodiment, the coupling moiety comprises additional atoms as a spacer. As will be appreciated by those in the art, a wide variety of groups may be used. For example, a coupling moiety may comprise an alkyl or aryl group substituted with one or more functional groups. Thus, in one embodiment, a coupling moiety containing a multiplicity of functional groups for attachment of multiple MRI contrast agents may be used, similar to the polymer embodiment described below. For example, branched alkyl groups containing multiple functional groups may be desirable in some embodiments.
In an additional embodiment, the linker is a polymer. In this embodiment, a polymer comprising at least one MRI contrast agent of the invention is used. Preferred embodiments utilize a plurality of MRI agents per polymer. The number of MRI agents per polymer will depend on the density of MRI agents per unit length and the length of the polymer.
The character of the polymer will vary, but what is important is that the polymer either contain or can be modified to contain functional groups for the the attachment of the MRI contrast agents of the invention. Suitable polymers include, but are not limited to, functionalized dextrans, styrene polymers, polyethylene and derivatives, polyanions including, but not limited to, polymers of heparin, polygalacturonic acid, mucin, nucleic acids and their analogs including those with modified ribose-phosphate backbones, the polypeptides polyglutamate and polyaspartate, as well as carboxylic acid, phosphoric acid, and sulfonic acid derivatives of synthetic polymers; and polycations, including but not limited to, synthetic polycations based on acrylamide and 2-acrylamido-2-methylpropanetrimethylamine, poly(N-ethyl-4-vinylpyridine) or similar quarternized polypyridine, diethylaminoethyl polymers and dextran conjugates, polymyxin B sulfate, lipopolyamines, poly(allylamines) such as the strong polycation poly(dimethyldiallylammonium chloride), polyethyleneimine, polybrene, spermine, spermidine and polypeptides such as protamine, the histone polypeptides, polylysine, polyarginine and polyornithine; and mixtures and derivatives of these. Particularly preferred polycations are polylysine and spermidine, with the former being especially preferred. Both optical isomers of polylysine can be used. The D isomer has the advantage of having long-term resistance to cellular proteases. The L isomer has the advantage of being more rapidly cleared from the subject. As will be appreciated by those in the art, linear and branched polymers may be used.
A preferred polymer is polylysine, as the —NH2 groups of the lysine side chains at high pH serve as strong nucleophiles for multiple attachment of activated chelating agents. At high pH the lysine monomers are coupled to the MRI agents under conditions that yield on average 5-20% monomer substitution.
In some embodiments, particularly when charged polymers are used, there may be a second polymer of opposite charge to the first that is electrostatically associated with the first polymer, to reduce the overall charge of polymer-MRI agent complex. This second polymer may or may not contain MRI agents.
The size of the polymer may vary substantially. For example, it is known that some nucleic acid vectors can deliver genes up to 100 kilobases in length, and artificial chromosomes (megabases) have been delivered to yeast. Therefore, there is no general size limit to the polymer. However, a preferred size for the polymer is from about 10 to about 50,000 monomer units, with from about 2000 to about 5000 being particularly preferred, and from about 3 to about 25 being especially preferred.
It should be understood that the multimeric MRI agents of the invention may be made in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the agents.
As outlined above, the MRI agents may also be combined into higher multimers, either by direct linkage or via attachment to a polymer.
The complexes of the invention are generally synthesized using well known techniques and as outlined in the Examples.
The contrast agents of the invention are complexed with the appropriate metal ion as is known in the art. While the structures depicted herein all comprise a metal ion, it is to be understood that the contrast agents of the invention need not have a metal ion present initially. Metal ions can be added to water in the form of an oxide or in the form of a halide and treated with an equimolar amount of a contrast agent composition. The contrast agent may be added as an aqueous solution or suspension. Dilute acid or base can be added if need to maintain a neutral pH. Heating at temperatures as high as 100░ C. may be required.
The complexes of the invention can be isolated and purified, for example using HPLC systems.
Pharmaceutical compositions comprising pharmaceutically acceptable salts of the contrast agents can also be prepared by using a base to neutralize the complexes while they are still in solution. Some of the complexes are formally uncharged and do not need counterions.
Once synthesized, the metal ion complexes of the invention have use as magnetic resonance imaging contrast or enhancement agents.
The metal ion complexes of the invention may be used in a similar manner to the known gadolinium MRI agents. See for example, Meyer et al., supra; U.S. Pat. Nos. 5,155,215; 5,087,440; Margerstadt et al., Magn. Reson. Med. 3:808 (1986); Runge et al., Radiology 166:835 (1988); and Bousquet et al., Radiology 166:693 (1988). The metal ion complexes are administered to a cell, tissue or patient as is known in the art. A "patient" for the purposes of the present invention includes both humans and other animals and organisms, such as experimental animals. Thus the methods are applicable to both human therapy and veterinary applications. In addition, the metal ion complexes of the invention may be used to image tissues or cells; for example, see Aguayo et al., Nature 322:190 (1986).
Generally, sterile aqueous solutions of the contrast agent complexes of the invention are administered to a patient in a variety of ways, including orally, intrathecally and especially intraveneously in concentrations of 0.003 to 1.0 molar, with dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram of body weight being preferred. Dosages may depend on the structures to be imaged. Suitable dosage levels for similar complexes are outlined in U.S. Pat. Nos. 4,885,363 and 5,358,704.
In addition, the contrast agents of the invention may be delivered via specialized delivery systems, for example, within liposomes (see Navon, Magn. Reson. Med. 3:876-880 (1986)) or microspheres, which may be selectively taken up by different organs (see U.S. Pat. No. 5,155,215).
In some embodiments, it may be desirable to increase the blood clearance times (or half-life) of the MRI agents of the invention. This has been done, for example, by adding carbohydrate polymers to the chelator (see U.S. Pat. No. 5,155,215). Thus, one embodiment utilizes polysaccharides as substitution R groups on the compositions of the invention.
A preferred embodiment utilizes complexes which cross the blood-brain barrier. Thus, as is known in the art, a DOTA derivative which has one of the carboxylic acids replaced by an alcohol to form a neutral DOTA derivative has been shown to cross the blood-brain barrier. Thus, for example, neutral complexes are designed that cross the blood-brain barrier with blocking moieties which detect Ca+2 ions. These compounds are used in MRI of a variety of neurological disorders, including Alzeheimer's disease. Currently it is difficult to correctly diagnosis Alzeheimer's disease, and it would be useful to be able to have a physiological basis to distinguish Alzeheimer's disease from depression, or other treatable clinical symptoms for example.
All references cited herein are incorporated by reference.
Below are given the reaction sequence for the cyclam (14aneN4) macrocyle. The same reaction sequence works identically for cyclen (14aneN4) and homocyclen (13aneN4). FIG. 3 depicts the synthetic scheme.
Diacetic Acid Bridged Cyclam:
In a 1 L roundbottom flask, 0.032 mol (7.248 g) H2 Bcyclam, synthesized according to reference 2, was stirred with 500 ml dry acetonitrile. Potassium carbonate, 35 g, was added and the vessel was placed under argon. Ethyl iodoacetate (2 eg., 13.70 g, 7.6 ml) was added and the reaction mixture stirred at room temperature for 3.5 h under argon. Upon reaction completion, the reaction mixture was filtered to remove K2CO3 and rotovapped to a solid. Mass Spec, and NMR characterization at this point were consistent with the diester. The ester groups were hydrolyzed to the acids at this point. The solid was dissolved in 400 ml 50% ethanol/water and stirred overnight with 300 ml IRA-400(OH.) anion exchange resin. [The resin had been prepared by washing with 2ū500 ml 2 M NaOH, then 500 ml water to remove excess OH−.] Filtration followed by solvent evaporation yielded 6.64 g (61%) of the product as a yellow viscous oil. Mass and NMR spectra were obtained in D2O and were consistent with the product.
Mn(AcBcyclam) and Mn(AcBcyclam)PF6 (Same for Fe2+/3+):
The AcBcyclam ligand (0.001 mol, 0.342 g) was suspended in 25 ml 4:1 acetonitrile:methanol solution in a 2-neck 100 ml roundbottom flask. A solid addition funnel was loaded with MnCl2 (0.001 mol, 0.126 g) and attached to the reaction flask. The solution was rigorously degassed by flushing it with argon then putting it under vacuum with the solvent boiled. This procedure was repeated four times. The reaction vessel was left under argon and the MnCl2 was added from the funnel. The pink MnCl2 dissolved quickly giving a gray suspension. The reaction was stirred at reflux for 1.5 h using a water bath (70░ C.). The reaction was then cooled in an ice bath to complete the precipitation, and the white suspended solid filtered on a fine glass frit. This solid was likely excess MnCl2. The pale green filtrate was evaporated to a pale orange sticky solid crude product. X-ray quality colorless crystals were grown from ether diffusion into a MeCN/MeOH solution. The complex in solution was air sensitive and grew more colored with time. Bulk recrystallization therefore did not yield pure product. ESMS+ gave large peaks at 396=MnL, and 434=MnLK.
Due to the air sensitivity of the product, it was oxidized to a more stable Mn(III) form as follows. 5 eq 30 NH4PF6 and the crude Mn(II) complex (6.32ū10−4 mol, 250 mg) were dissolved in 12 ml MeOH. Argon was bubbled through the solution for 5 minutes. Molecular bromine (5 drops) was added to the reaction mixture causing an instant precipitation of an orange solid. Argon was again bubbled through the reaction (15 min) to remove excess Br2. The reaction was cooled at 4░ C. overnight to complete precipitation. The product was then collected on a fine glass frit and washed with MeOH and ether. The yield was 283 mg. ESMS+ gave a large peak at 395=MnL. Elemental analysis calc for MnLPF6.NH4PF6.MeOH: C 27.77, H 4.93, N 9.52. Found: C 27.93, H 4.41, N 907.
Gd(AcBcyclam)CI and Gd(AcBcyclam)PF6:
The ligand AcBcyclam (0.001 mol, 0.342 g) was dissolved in 15 dry DMF and placed under argon. To this stirring solution was added GdCl3 (0.001 mol, 0.264 g) as a solid. The reaction mixture was heated to reflux under argon overnight. A white suspended solid was present in the reaction mixture. Attempts at filtration of this material (KCl?) were unsuccessful, so all materials were recombined by redissolving any solid with MeOH. The combined DMF/MeOH solution was evaporated to an off-white solid. Electrospray mass spec. gave peaks at 534=GdLCl and 572=KGdLCl. Ether diffusion into a methanol solution gave colorless crystals whose X-ray structure has been determined and matches that given below. Elemental analysis: calc GdLCl.KCl.4H2O: C 27.54, H 5.49, N 8.03; found C 27.70, H 4.87, N 8.20. The relaxivity of the chloride complex was measured at 22░ C. at 500 Mgz, at pH 5.5 and was found to be 7.5 mM−1s−1, roughly twice that of currently clinically used contrast agents.
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