text:
From Chapter I., The Milesian School
15. Aristotle's Account of the Theory
In a number of other places Aristotle speaks of some one who held the primary substance to be something "intermediate between" the elements or between two of them .62 Nearly all the Greek commentators referred this to Anaximander also, but most modern writers refuse to follow them. It is, no doubt, easy to show that Anaximander himself cannot have said anything of the sort, but that is no real objection. Aristotle puts things in his own way regardless of historical considerations, and it is difficult to see that it is more of an anachronism to call the Boundless "intermediate between the elements" than to say that it is "distinct from the elements." Indeed, if once we introduce the elements at all, the former description is the more adequate of the two. At any rate, if we refuse to understand these passages as referring to Anaximander, we shall have to say that Aristotle paid a great deal of attention to some one whose very name has been lost, and who not only agreed with some of Anaximander's views, but also used some of his most characteristic expressions.63 We may add that in one or two places Aristotle certainly seems to identify the "intermediate" with the something "distinct from" the elements.64
There is even one passage in which he speaks of Anaximander's Boundless as a "mixture," though his
words may perhaps admit of another interpretation.65 But this is of no consequence for our
interpretation of Anaximander. It is certain that he cannot have said anything about "elements," which no
one thought of before Empedokles, and no one could think of before Parmenides. The question has
only been mentioned because it has given rise to a lengthy controversy, and because it throws light on
the historical value of Aristotle's statements. From the point of view of his own system, these may be
justified; but we shall have to remember in other cases that, when he seems to attribute an idea to some
earlier thinker, we are not bound to take what he says in an historical sense.66
Burnet's Notes
60. Arist. Met. Λ, 2. 1069 b 18 (R. P. 16 c).
61. This is taken for granted in Phys. Γ, 4. 203 a 16; 204 b 22 (R. P. 16 b), and stated in Γ, 8. 208 a 8 (R. P. 16 a). Cf. Simpl. Phys. p. 150, 20 (R. P. 18).
62. Aristotle speaks four times of something intermediate between Fire and Air (Gen. Corr. B, 1. 328 b 35; ib. 5. 332 a 21; Phys. A, 4. 187 a 14; Met. A, 7. 988 a 30). In five places we have something intermediate between Water and Air (Met. A, 7. 988 a 13; Gen. Corr. B, 5. 332 a 21; Phys. Γ, 4. 203 a 18; ib. 5. 205 a 27; De caelo, Γ, 5. 303 b 12). Once (Phys. A, 6. 189 b 1) we hear of something between Water and Fire. This variation shows at once that he is not speaking historically. If any one ever held the doctrine of τὸ μεταξύ, he must have known which "elements" he meant.
63. Arist. De caelo, Γ, 5. 303 b 12, ὕδατος μὲν λεπτότερον, ἀέρος δὲ πυκνότερον, ὃ περιέχειν φασὶ πάντας τοὺς οὐρανοὺς ἄπειρον ὄν.
64. cf. Phys. Γ, 5. 204 b 22 (R. P. 16 b), where Zeller rightly refers τὸ παρὰ τὰ στοιχεῖα to Anaximander. Now, at the end (205 a 25) the whole passage is summarised thus: καὶ διὰ τοῦτ' οὐθεὶς τὸ ἓν καὶ ἄπειρον πῦρ ἐποίησεν οὐδὲ γῆν τῶν φυσιολόγων, ἀλλ' ἢ ὕδωρ ἢ ἀέρα ἢ τὸ μέσον αὐτῶν. In Gen. Corr. B, 1. 328 b 35 we have first τι μεταξὺ τούτων σῶμά τε ὂν καὶ χωριστόν, and a little further on (329 a 9) μίαν ὕλην παρὰ τὰ εἰρημένα. In B, 5. 332 a 20 we have οὐ μὴν οὐδ' ἄλλο τί γε παρὰ ταῦτα, οἷον μέσον τι ἀέρος καὶ ὕδατος ἢ ἀέρος καὶ πυρός.
65. Met. Λ, 2. 1069 b 18 (R. P. 16 c). Zeller (p. 205, n. 1) assumes an "easy zeugma."
66. For the literature of this controversy, see R. P. 15. Professor Heidel has shown in his "Qualitative Change in Pre-Socratic Philosophy" (Arch., xix. p. 333) that Aristotle misunderstood the Milesians because he could only think of their doctrine in terms of his own theory of ἀλλοίωσις. That is quite true, but it is equally true that they had no definite theory of their own with regard to the transformations of substance. The theory of an original "mixture" is quite as unhistorical as that of ἀλλοίωσις. Qualities were not yet distinguished from "things," and Thales doubtless said that water turned into vapour or ice without dreaming of any further questions. They all believed that in the long run there was only one "thing," and at last they came to the conclusion that all apparent differences were due to rarefaction and condensation. Theophrastos (ap. Simpl. Phys. 150, 22) says ἐνούσας γὰρ τὰς ἐναντιότας ἐν τῷ ὑποκειμένῳ . . . ἐκκρίνεσθαι. I do not believe these words are even a paraphrase of anything Anaximander said. They are merely an attempt to "accommodate" his views to Peripatetic ideas, and ἐνούσας is as unhistorical as the ὑποκείμενον.
Created for Peithô's Web from Early Greek Philosophy by John Burnet, 3rd edition (1920). London: A & C Black Ltd. Burnet's footnotes have been converted to chapter endnotes. Greek unicode text entered with Peithô's Younicoder.
Web design by Larry Clark and RSBoyes (Agathon). Peithô's Web gratefully acknowledges the assistance of Anthony Beavers in the creation of this web edition of Burnet. Please send comments to:


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Butterick 8859
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Is this restaurant family-friendly?
Does this restaurant have tables with seating?
Is this restaurant good for lunch?
Is this a Mediterranean restaurant?
Does this restaurant offer highchairs for toddlers?
Does this restaurant offer outdoor seating?
Is this restaurant romantic?
Is this restaurant good for business meetings?
Is this restaurant good for bar scene?
Is this a Turkish restaurant?
Thanks for helping!
.....spent vegging out here, reading our Kindles, swimming in the lagoon with the fish, eating lovely food in the restaurant and watching the staff prepare for evening weddings. Oh- and watching the hang gliders overhead from Babadag mountain.
Spent a day at Seahorse recently and had lunch there, the food was great , good potions and good value, would recommend, have been to other beaches nearby but this is by far the best.
Tried a few of the beaches on the lagoon and tried seahorse this year. It's about the norm for the area. Food is reasonable , had a few lunches there and have no complaints. Costs 20tl each for sunbed and parasol. Free shuttle bus to and from your hotel. Worth a visit.
paying 20 tl for this place is terrible value for money, but beware when you order food they will give you anything available on my english breky was 1 beefburger with stail bap,chips,egg(hard) and 1 bacon. (menu) beans,sausage,bacon,egg,b and b. I would have eaten it if it wasnt so bad. After complaint he got me a beer.
Surprised by the good reviews here. We thought the beach area wasnt great, sand was coarse and like builders cement. Loungers far too close together. Just wasnt impressed by any if it. Stayed under two hours bjt would have left immediately if i could.
Really nice beach club, bit less busy than the others around the area, food was lush, good facilities and bus picks you up from hotel and they have schedule for times for drop off or you can walk if you feel like a stroll. Spent a couple of really nice days here
A lovely place at the end of olu deniz in the bay. We went to the restaurant in the evening for dinner. Could hear the crickets and had a visit from the kittens. It's very tranquil setting and the restaurant is decorated very nicely - wooden beams, floors benches and chairs. The food was great and the owner Yasin is...
This place never fails to deliver. The beach is clean and it is in a great location on the lagoon. The restaurant serves excellent food and the staff are friendly. Would recommend you to take a trip around the lagoon on the pedalos - this time we saw three loggerhead turtles in a one hour trip.
Spent a enjoyable day at the Sea Horse Beach Club. A 20 lira entrance fee p/p included pick up and return to our hotel and use of sunbeds and parasol. Had a meal during lunch time - food was very good and not over priced. Highlight of the day was following a couple of turtles around in a peddleo ....
A simple small sandy beach by the lovely calm blue lagoon (owned by Thomson holidays-I think) for couples and families. Great restaurant-quality food at reasonable rates, quiet areas, clean loos and the sunset suppers are very good and highly recommended.
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Presumably not happy with the results of previous consultations, Lambeth Council are now opening up another dialogue with people who use Brixton Rec as well as local residents and organisations.
The council is asking for feedback about the activities and services provided at the Rec, and wants to know, "how we can make it a better place for everyone to use."
Working with the Brixton Rec User Group and GLL (Greenwich Leisure Limited – the company running the centre for the council), Lambeth want to know, "what people have to say about activities and services available at the centre, what you would like to see more of, and other things that you would like to see improved – like equipment and facilities."
There will be a public meeting at the Brixton Recreation Centre on Monday 12 May 2014, 7 to 8.30pm, and they are inviting users to fill in a questionnaire:
Brixton Rec questionnaire long version – Brixton Rec questionnaire short version.
Join the discussion on urban75 about the Brixton Rec consultation.


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Torque XLT TQ T29
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The rear ramp door allows for easy loading and unload of your toys and gear. There is 12' of garage space which is a nice area for all of your off-road toys. After everything is unloaded you can set up the bench seat during the day for more seating and at night there is an electric queen bed and the bench seat makes into a bed for more sleeping space. In the garage area you will also find a side door which allows easy access to the inside of the trailer without opening the rear door.
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The kitchen area has a refrigerator, double kitchen sink, three burner range, extra counter space and an overhead cabinet.
Proceeding toward the front of the trailer, the bathroom is on the left side. Inside the bathroom there is a toilet, linen cabinet for towels or toiletries, corner shower, sink, and medicine cabinet. The bathroom also has a door which leads directly into the bedroom.
In the front bedroom you will find a king bed with nightstands and storage above, and more!We have 1 TQ T29 availableView Inventory
Sleeps 7 Slides 1 Length 34 ft 5 in Ext Width 8 ft 6 in Ext Height 12 ft 7 in Hitch Weight 1355 lbs Gross Weight 9999 lbs Dry Weight 7785 lbs Fresh Water Capacity 98 gals Grey Water Capacity 63 gals Black Water Capacity 63 gals Tire Size ST235/80R16 LR E
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text:
One must accomplish two things when earning a PhD. First one must understand a particular subject thoroughly. Secondly, one must extensively in knowledge about that subject.
Spanning 23 countries, North America is continent filled with educational opportunity. Students have the opportunity to learn multiple languages and develop an understanding of vastly different cultures.
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The Doctor of Philosophy (PhD) in Religious Studies is an advanced research degree leading to a dissertation. Research and writing skills are developed to the level necessary to participate in professional academic discourse. A doctoral degree is awarded on the basis of evidence that the recipient possesses knowledge of a broad field of learning and expert mastery of a particular area of concentration within that field. [+]
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Be equipped for greater ministry effectiveness. Our Doctor of Ministry invites men and women to step deeper into study of the Word, to broaden their perspectives on ministry practices, and to join with a global community of ministry practitioners in dialogue and study for the sake of the Gospel. [+]


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Heidi E. Murkoff
With over 10.5 million copies in print, What to Expect the First Year is the world's best-selling, best-loved guide for parents. Now, it's better ...
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I bought this book for our daughter and her husband.
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This is a must have for expectant parents.
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SpringHill Suites by Marriott Fairfax Fair Oaks
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(703) 691-7880
11191 Waples Mill Rd, Fairfax, VA 22030
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Property Location Located in Fairfax, SpringHill Suites by Marriott Fairfax Fair Oaks is convenient to National Firearms Museum and Fair Oaks Mall. This hotel is within close proximity of Penderbrook Golf Club and Ox Hill Battlefield Park.
Rooms Make yourself at home in one of the 140 air-conditioned rooms featuring refrigerators and microwaves. Wired and wireless Internet access is complimentary, while 32-inch flat-screen televisions with satellite programming provide entertainment. Private bathrooms with bathtubs feature complimentary toiletries and hair dryers. Conveniences include desks and separate sitting areas, and housekeeping is provided daily.
Amenities Take advantage of recreation opportunities including an indoor pool and a fitness center. Additional amenities at this hotel include complimentary wireless Internet access, gift shops/newsstands, and wedding services.
Dining A complimentary buffet breakfast is served daily.
Business, Other Amenities Featured amenities include complimentary high-speed (wired) Internet access, a 24-hour business center, and express check-out. Planning an event in Fairfax? This hotel has facilities measuring 780 square feet (72 square meters), including a meeting room. Free self parking is available onsite.
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Need a book? Engineering books recommendations...
Return to index: [Subject] [Thread] [Date] [Author]
Re: Caisson cold joint[Subject Prev][Subject Next][Thread Prev][Thread Next]
- To: seaint(--nospam--at)seaint.org
- Subject: Re: Caisson cold joint
- From: Roger Davis <sds_rdavis(--nospam--at)yahoo.com>
- Date: Fri, 8 Dec 2006 05:51:10 -0800 (PST)
ACI 336.1 says that uncased or bell excavated drilled piers are to be completely placed during one working day. It also says that cased piers must be placed in one continuour operation unless directed by the Owner's rep. If there is a construction joint it needs to be treated like a construction joint in a column or beam. It says that dowels or shear key should be provided when directed by the Owner's rep. The caissons will have to act as beam-columns to support the lights so they should be treated pretty much like beam-columns above grade.
Roger Davis
SDS Architects, Inc.
Jim Wilson <wilsonengineers(--nospam--at)yahoo.com> wrote:
Jim Wilson <wilsonengineers(--nospam--at)yahoo.com> wrote:
100ft tall light poles are being erected on 4ft diameter caisson's at a local university. The contractor has asked if he can place a cold joint down about 4ft below the top of the caisson. This will facilitate installation of the anchor bolts and templates as well as electrical conduits.Is this generally acceptable and is it common? The Engineer for the pole manufacturer designed the foundation, but they have not been asked about the cold joint. Maybe that answer needs to come from the owner's engineer, but they are electical and not qualified to answer the question. So they in turn asked me but I know very little about caissons and told them I would at least ask the appropriate people.Thanks in advance for any advice on this.Jim Wilson, PEStroudsburg, PA
Access over 1 million songs - Yahoo! Music Unlimited.Roger DavisArchitectSDS Architects, Inc.
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In an interview with Billy Bush on Access Hollywood, Jay Leno explains why he never had Joan Rivers on NBC's Tonight Show. He says the show didn't want to disrespect Johnny Carson by inviting Joan, who had a long time beef with Carson, onto the show. http://goo.gl/WtAkMG #EntertainmentBiz
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Captain marvel shazam
Explore related topics
youngjusticer: Billy Batson was an orphan chosen to be a champion of good by a wizard who gifted Batson with the power of six legendary Greek figures, and when he spoke the wizard's name, he became an adult hero empowered by gods. Check out Flash and Green Lantern as well. SHAZAM! Captain Marvel, by Kim Intae.
Don't love the poster so much but at least I have something to remind me what Shazam actually stands for...
Shazam by Alex Ross
Absolutely one of my favorite super heroes
Captain Marvel by Alex Ross
Freddy Freeman/Captain Marvel Jr./Shazam/Powers-Super-Strength, Super-Speed, Invulnerability, Flight, Healing, Wisdom, Enhanced Mental Perception, Fearlessness, Magic Lightning
Captain Marvel's new "Shazam!" interpretation by DC Comics. I might have to start collecting this one.
Shazam (=^.^=) Thanks, Pinterest Pinners, for stopping by, viewing, re-pinning, & following my boards. Have a beautiful day! ^..^ and "Feel free to share on Pinterest ^..^ #comicbookheros #unocollectibles
Captain Marvel - Mighty by Steve Garcia
Superman finds a foe who may be too powerful even for him to defeat in the murderous Black Adam. Fortunately, Black Adam's real target is the Earth's Mightiest Mortal--Captain Marvel.


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You were about to say there's something else in the world you fear most?
Yeah, I didn't think so.
What makes a good zombie movie? It's all a matter of opinion. I'm not going to give you a top ten list telling you the best and the worst ones out there. This is more of a zombie appreciation post. I love me a good zombie flick. Whether it's for laughs or to try to scare you with how "realistic" it could be, they are always entertaining.
This guy will make you laugh. Har har har.
In my opinion, a good zombie movie has something for everyone. The all around tough theme of zombie apocalypse, some lighthearted humor, the perfect balance of blood and guts, a wicked soundtrack and maybe a little romance.
Show me some zombie love!
When it comes to zombie movies, though, my main concern is all about creativity. With a million different zombie movies out there, you've seen it all. All the reasons why zombies would suddenly take over the planet have been done and redone to death (ha). But if I'm investing my (sort of) valuable time, I want to see something interesting. Explain this new disease to me. Why is life on earth coming to an end? How did it all start? Come up with something good! Something we haven't heard before. Or if we have, polish it up and show it to us from a new point of view.
Or, if you're going to go the rout of not giving the audience any sort of reasoning why someone is trying to eat my face off, put that creativity to use elsewhere. Put those zombies and their victims in an unusual location. Give me some interesting character development. Don't just give the actors an unlimited access to guns and ammo. Let's see them figure something else out!
That could work.
I'd love to see some more development with the undead. Not many people pay attention to it, but the next time you're watching one of these Oscar worthy films, take some note to how fast it takes these folks to turn into zombies. Is it instantly? Are they dead for a few minutes to give their loved ones some time to grieve?
Shoulda seen that coming.
More details that I love about zombie movies is the actual zombie. How do they look? Good? Nah. They look like shit. But filmmakers, come on, give us a good close up look at 'em. There are some amazing makeup artists out there and I want to see what they can do! I want to see what your idea of a zombie is going to look like.
That's a face only a mother could love.
And are you going for a humorous zombie film or to try to scare the pee out of me? I'd like to know ahead of time so I could prepare myself with the right underwear.
I can't think of anything scarier than this face. Just looking at it now I feel tears welling up and want to cry out for my momma.
And how are you going to achieve that perfect mix of all the great things that make up a good zombie movie? I personally think there are too many horror films out there today that try too hard for the gross-out effect than anything else. I lose a little respect for a film if all I see are insides on the outside and faces being blown to pieces every five minutes. I mean, there's a time and a place, but we need a break from the nasty stuff every once in a while in order to really appreciate it.
Ooh that stings!
And can we stop trying to remake the classics? Unless it's Romero saying he wants to be involved in the remake of one of his own movies, don't touch his stuff. That goes for all the rest. Come up with your own ideas and see those through before you even think about rewriting the script of an oldie but a goodie. I see this with too many movies of all genres already. It's one thing to get ideas from other movies, but don't you dare try to fix something that ain't broken!
To conclude, I will leave you with this: My zombie film will be completed and will blow your minds. I'm not sure when, but it will. I only hope that it's on the big screen sometime before zombies really do take over.
Happy zombie hunting!


text:
|Publication number
|Publication type
|Application number
|Publication date
|Nov 9, 1999
|Filing date
|Mar 25, 1998
|Priority date
|Mar 31, 1997
|Fee status
|Also published as
|Publication number
|James Arthur Hoffmann
|Original Assignee
|Eli Lillly And Company
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (11), Non-Patent Citations (41), Referenced by (80), Classifications (14), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
R1 -Met-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R2 (SEQ ID NO: 2)
This application claims the benefit of U.S. Provisional Application Ser. No. 60/042,167, filed Mar. 31, 1997.
The present invention relates to protein chemistry as applied to pharmaceutical research and development. The invention provides novel peptides and compositions that are useful for treating diabetes.
The World Health Organization estimates that there may be as many of 103 million patients with type II diabetes worldwide, although only 23 million have been diagnosed and are receiving therapy. Type II diabetes (non-insulin dependent diabetes mellitus--NIDDM) is characterized by a resistance to insulin action in peripheral tissues such as muscle, adipose and liver and by a progressive failure in the ability of the islet β-cell to secrete insulin. Because current therapeutics do not halt the progression of β-cell failure, virtually all NIDDM patients eventually require insulin to control blood glucose levels. The most commonly described therapeutics for such patients are the sulfonylureas, so called oral agents, that stimulate insulin secretion. Each year, 10-20% of the patients on sulfonylureas fail to maintain acceptable blood glucose levels and switch to insulin therapy.
Insulin however, is a difficult drug for patients to self-administer for several reasons. First, insulin has a narrow therapeutic index. This leads to poor control of blood glucose levels since most patients and physicians prefer elevated glucose levels to the risk of hypoglycemia and coma. Second, proper insulin dosing is complicated. The insulin dose that a diabetic patient should administer is dependent on the amount of food consumed, the time between meals, the amount of physical exercise, and the prevailing blood glucose level which requires blood glucose monitoring to determine. The general diabetic population is ill-equipped to correlate these factors to the proper insulin dose. Third, as a parenteral product, insulin is inconvenient to administer. Alternative delivery methods are made even more difficult by the narrow therapeutic index for insulin. Thus, many diabetic patients lack good control of blood glucose.
The Diabetes Control and Complication Trial definitively established for Type I diabetics that disease complications (retinopathy, neuropathy and nephropathy) are directly correlated to blood glucose control. A clinical trial is currently underway in the UK to determine if this link also holds true to Type II diabetes. A positive result is reasonable to anticipate, and with it will come a desire for agents that improve the ability for the patient with diabetes to control blood glucose levels tightly. In addition, a standard of care for diabetes is being developed by the US government in coordination with care-givers and the pharmaceutical industry. Thus, there is clearly a need for agents that truly are able to tightly control blood glucose levels in the normal range.
Glucagon-like peptide-1 (GLP-1) was first identified in 1987 as a incretin hormone, a peptide secreted by the gut upon ingestion of food. GLP-1 is secreted by the L-cells of the intestine after being proteolytically processed from the 160 amino acid precursor protein, preproglucagon. Cleavage of preproglucagon first yields GLP-1, a 37 amino acid peptide, GLP-1(1-37)OH, that is poorly active. A subsequent cleavage at the 7-position yields biologically active GLP-1(7-37)OH. Approximately 80% of the GLP-1(7-37)OH that is synthesized is amidated at the C-terminal after removal of the terminal glycine residue in the L-cells. The biological effects and metabolic turnover of the free acid GLP-1(7-37)OH, and the amide, GLP-1 (7-36)NH2, are indistinguishable.
GLP-1 is known to stimulate insulin secretion (insulinotropic action) causing glucose uptake by cells which decreases serum glucose levels (see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)). Numerous GLP-1 analogs demonstrating insulinotropic action are known in the art. These variants and analogs include, for example, GLP-1(7-36), Gln9 -GLP-1(7-37), D-Gln9 -GLP-1(7-37), acetyl-Lys9 -GLP-1(7-37), Thr16 -Lys18 -GLP-1(7-37), and Lys18 -GLP-1(7-37). Derivatives of GLP-1 include, for example, acid addition salts, carboxylate salts, lower alkyl esters, and amides (see, e.g., WO91/11457 (1991); EP 0 733,644 (1996); and U.S. Pat. No. 5,512,549 (1996)). It has also been demonstrated that the N-terminal histidine residue (His 7) is very important to insulinotropic activity of GLP-1 (Suzuki, S., et al. Diabetes Res.; Clinical Practice 5 (Supp. 1):S30 (1988).
Multiple authors have demonstrated the nexus between laboratory experimentation and mammalian, particularly human, insulinotropic responses to exogenous administration of GLP-1, particularly GLP-1 (7-36)NH2 and GLP-1 (7-37) [see, e.g., Nauck, M. A., et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., New England J. of Medicine, 326(20):1316-1322 (1992); Nauck, M. A., et al., J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes, 42:1219-1225 (1993)].
GLP-1 based peptides hold great promise as alternatives to insulin therapy for patients with diabetes who have failed on sulfonylureas. GLP-1 has been studied intensively by academic investigators, and this research has established the following for patients with type II diabetes who have failed on sulfonylureas:
1) GLP-1 stimulates insulin secretion, but only during periods of hyperglycemia. The safety of GLP-1 compared to insulin is enhanced by this property of GLP-1 and by the observation that the amount of insulin secreted is proportional to the magnitude of the hyperglycemia. In addition, GLP-1 therapy will result in pancreatic release of insulin and first-pass insulin action at the liver. This results in lower circulating levels of insulin in the periphery compared to subcutaneous insulin injections. 2) GLP-1 suppresses glucagon secretion, and this, in addition to the delivery of insulin via the portal vein helps suppress the excessive hepatic glucose output in diabetic patients. 3) GLP-1 slows gastric emptying which is desirable in that it spreads nutrient absorption over a longer time period, decreasing the postprandial glucose peak. 4) Several reports have suggested that GLP-1 may enhance insulin sensitivity in peripheral tissues such as muscle and fat. 5) Finally, GLP-1 has been shown to be a potential regulator of appetite.
Meal-time use of GLP-1 based peptides offers several advantages over insulin therapy. Insulin therapy requires blood glucose monitoring, which is both expensive and painful. The glucose-dependency of GLP-1 provides an enhanced therapeutic window in comparison to insulin, and should minimize the need to monitor blood glucose. Weight gain also can be a problem with intensive insulin therapy, particularly in the obese type II diabetic patients.
The therapeutic potential for native GLP-1 is further increased if one considers its use in patients with type I diabetes. A number of studies have demonstrated the effectiveness of native GLP-1 in the treatment of insulin dependent diabetes mellitus (IDDM). Similar to NIDDM patients, GLP-1 is effective in reducing fasting hyperglycemia through its glucagonostatic properties. Additional studies have indicated that GLP-1 also reduces postprandial glycemic excursions in IDDM, most likely through a delay in gastric emptying. These observations indicate that GLP-1 is may be useful as a treatment in IDDM as well as in NIDDM.
However, the biological half-life of native GLP-1 molecules which are affected by the activity of dipeptidyl-peptidase IV (DPP IV) is quite short. For example, the biological half-life of GLP-1(7-37)OH is a mere 3 to 5 minutes (U.S. Pat. No. 5,118,666). Therefore extended-action GLP-1 based peptides are needed to enhance glycemic control during the night while minimizing the risk of hypoglycemia.
In one embodiment, the present invention provides GLP-1 analogs that have extended time action relative to native GLP-1, show resistance to the action of DPP-IV, and retain affinity for the GLP-1 receptor.
The invention includes a GLP-1 compound of the formula:
R1 -X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-val-Lys-Gly-Arg-R2 (SEQ ID NO:1)
or a pharmacuetically accetable salt thereof, wherein:
R1 is selected from the group consisting of His, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;
X is selected from the group consisting of Met, Asp, Lys, Thr, Leu, Asn, Gln, Phe, Val, and Tyr
Y and Z are independently selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly, and;
R2 is selected from the group consisting of NH2, and Gly-OH; provided that, if R1 is His, X is Val, Y is Glu, and Z is Glu, then R2 is NH2.
Also provided by the present invention are pharmaceutical compositions comprising a GLP-1 compound of the present invention in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.
The present invention further provides a method of treating diabetes which comprises administering to a mammal in need of such treatment an effective amount of a GLP-1 compound of the present invention.
In one embodiment, the present invention provides novel, biologically-active GLP-1 based peptides. It should be noted that this specification uses the nomenclature scheme that has developed around processed forms of GLP-1. In this scheme, the amino terminus of native GLP-1(7-37)OH has been assigned number 7 and the carboxy terminus number 37. Therefore, R1 of SEQ ID NO: 1 corresponds to residue 7 of GLP-1(7-37)OH. Likewise X in SEQ ID NO: 1 corresponds to residue 8 of GLP-1(7-37)OH and Y corresponds to residue 21 and so forth. Moreover, all amino acids referred to in this specification are in the L form, unless otherwise specified.
In a preferred embodiment, R1 is His, and Y and Z are Glu. Another preferred group is when R1 is His, R2 is Gly-OH, and any one or more of X, Y, and Z differ from the residues present in native GLP-1(7-37)OH. Another preferred group is when R1 is His, X is Met, Asp, Lys, Thr, Leu, Asn, Gln, Phe or Typ, Y and Z are Glu, and R2 is Gly-OH.
Given the sequence information herein disclosed and the state of the art in solid phase protein synthesis, GLP-1 analogs can be obtained via chemical synthesis. However, it also is possible to obtain a GLP-1 analog by fragmenting proglucagon using, for example, proteolytic enzymes. Moreover, recombinant DNA techniques may be used to express GLP-1 analogs of the invention.
The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Dugas, H. and Penney, C., Bioorganic Chemistry (1981) Springer-Verlag, New York, pgs. 54-92, Merrifield, J. M., Chem. Soc., 85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco, 1969).
For example, a GLP-1 analog of the invention may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404) and synthesis cycles supplied by Applied Biosystems. Boc amino acids and other reagents are commercially available from Applied Biosystems and other chemical supply houses. Sequential Boc chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides. For the production of C-terminal acids, the corresponding PAM resin is used. Asp, Gln, and Arg are coupled using preformed hydroxy benzotriazole esters. The following side chain protecting groups may be used:
Arg, Tosyl
Asp, cyclotlexyl
Glu, cyclohexyl
Ser, Benzyl
Thr, Benzyl
Tyr, 4-bromo carbobenzoxy
Boc deprotection may be accomplished with trifluoroacetic acid in methylene chloride. Following completion of the synthesis the peptides may be deprotected and cleaved from the resin with anhydrous hydrogen fluoride (HF) containing 10% meta-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at zero degrees centigrade or below, preferably -20� C. for thirty minutes followed by thirty minutes at 0� C. After removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and lyophilized.
The preparation of protected, unprotected, and partially protected GLP-1 has been described in the art. See U.S. Pat. Nos. 5,120,712 and 5,118,666, herein incorporated by reference, and Orskov, C., et al., J. Biol. Chem., 264 (22):12826-12829 (1989) and WO 91/11457 (Buckley, D. I., et al., published Aug. 8, 1991).
Likewise, the state of the art in molecular biology provides the ordinarily skilled artisan another means by which GLP-1 analogs can be obtained. Although GLP-1 analogs may be produced by solid phase peptide synthesis, recombinant methods, or by fragmenting glucagon, recombinant methods may be preferable because higher yields are possible. The basic steps in the recombinant production of a GLP-1 analog are:
a) isolating a natural DNA sequence encoding GLP-1 or constructing a synthetic or semi-synthetic DNA coding sequence for GLP-1,
b) placing the coding sequence into an expression vector in a manner suitable for expressing proteins either alone or as a fusion proteins,
c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector,
d) culturing the transformed host cell under conditions that will permit expression of a GLP-1 intermediate, and
e) recovering and purifying the recombinantly produced protein.
As previously stated, the coding sequences for GLP-1 analogs may be wholly synthetic or the result of modifications to the larger, native glucagon-encoding DNA. A DNA sequence that encodes preproglucagon is presented in Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982) and may be used as starting material in the recombinant production of a GLP-1 analog by altering the native sequence to achieve the desired results.
Synthetic genes, the in vitro or in vivo transcription and translation of which results in the production of a GLP-1 analog, may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode GLP-1 intermediates.
The methodology of synthetic gene construction is well known in the art. See Brown, et al. (1979) Methods in Enzymology, Academic Press, N.Y., Vol. 68, pgs. 109-151. DNA sequences that encode GLP-1 intermediates can be designed based on the amino acid sequences herein disclosed. Once designed, the sequence itself may be generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404).
To effect the expression of a biologically-active GPL-1 analog, one inserts the engineered synthetic DNA sequence in any one of many appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases. See generally Maniatis et al. (1989) Molecular Cloning; A Laboratory Manual, Cold Springs Harbor Laboratory Press, N.Y., Vol. 1-3. Restriction endonuclease cleavage sites are engineered into either end of the DNA encoding the GLP-1 analog to facilitate isolation from, and integration into, known amplification and expression vectors. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The choice of restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the protein of interest. The coding sequence must be positioned so as to be in proper reading frame with the promoter and ribosome binding site of the expression vector, both of which are functional in the host cell in which the protein is to be expressed.
To achieve efficient transcription of the coding region, it must be operably associated with a promoter-operator region. Therefore, the promoter-operator region of the gene is placed in the same sequential orientation with respect to the ATG start codon of the coding region.
A variety of expression vectors useful for transforming prokaryotic and eukaryotic cells are well known in the art. See The Promega Biological Research Products Catalogue (1992) (Promega Corp., 2800 Woods Hollow Road, Madison, Wis., 53711-5399); and The Stratagene Cloning Systems Catalogue (1992) (Stratagene Corp., 11011 North Torrey Pines Road, La Jolla, Calif., 92037). Also, U.S. Pat. No. 4,710,473 describes circular DNA plasmid transformation vectors useful for expression of exogenous genes in E. coli at high levels. These plasmids are useful as transformation vectors in recombinant DNA procedures and:
(a) confer on the plasmid the capacity for autonomous replication in a host cell;
(b) control autonomous plasmid replication in relation to the temperature at which host cell cultures are maintained;
(c) stabilize maintenance of the plasmid in host cell populations;
(d) direct synthesis of a protein prod. indicative of plasmid maintenance in a host cell population;
(e) provide in series restriction endonuclease recognition sites unique to the plasmid; and
(f) terminate mRNA transcription.
These circular DNA plasmids are useful as vectors in recombinant DNA procedures for securing high levels of expression of exogenous genes.
Having constructed an expression vector for a GLP-1 analog, the next step is to place the vector into a suitable cell and thereby construct a recombinant host cell useful for expressing a GLP-1 analog. Techniques for transforming cells with recombinant DNA vectors are well known in the art and may be found in such general references as Maniatis, et al. supra. Host cells made be constructed from either eukaryotic or prokaryotic cells. Eukaryotic host cells are capable of carrying out post-translational glycosylations on expressed proteins and some are capable of secreting the desired protein into the culture medium.
Prokaryotic host cells generally produce the protein at higher rates, are easier to culture but are not capable of glycosylating the final protein. Proteins which are expressed in high-level bacterial expression systems may aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Such protein aggregates must be solubilized, denatured and refolded using techniques well known in the art. See Kreuger, et al. (1990) in Protein Folding, Gierasch and King, eds., pgs 136-142, American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C.; and U.S. Pat. No. 4,923,967.
Regardless of the methods used to produce a GLP-1 analog, purification of the protein generally will be required. Methods for purifying proteins are well known in the art and include conventional chromatography, including ion and cation exchange, hydrophobic interaction, and immuno-affinity chromatographic media. The amino acid sequences herein disclosed in conjunction with well known protein purification methods will enable the ordinarily skilled artisan to purify GLP-1 analogs claimed herein.
The present invention also includes salt forms of GLP-1 analogs. A GLP-1 analog of the invention may be sufficiently acidic or sufficiently basic to react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and, especially, hydrochloric acid.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like. Salt forms of GLP-1 analogs are particularly preferred. Of course, when the compounds of this invention are used for therapeutic purposes, those compounds may also be in the form of a salt, but the salt must be pharmaceutically acceptable.
The ability of a GLP-1 analog to stimulate insulin secretion may be determined by providing a GLP-1 analog to cultured animal cells, such as the RIN-38 rat insulinoma cell line, and monitoring the release of immunoreactive insulin (IRI) into the media. Alternatively one can inject a GLP-1 analog into an animal and monitor plasma levels of immunoreactive insulin (IRI).
The presence of IRI is detected through the use of a radioimmunoassay which can specifically detect insulin. Any radioimmunoassay capable of detecting the presence of IRI may be employed; one such assay is a modification of the method of Albano, J. D. M., et al., Acta Endocrinol., 70:487-509 (1972). In this modification, a phosphate/albumin buffer with a pH of 7.4 is employed. The incubation is prepared with the consecutive addition of 500 μl of phosphate buffer, 50 μl of perfusate sample or rat insulin standard in perfusate, 100 μl of anti-insulin antiserum (Wellcome Laboratories; 1:40,000 dilution), and 100 μl of [125 I) insulin, giving a total volume of 750 μl in a 10�75 mm disposable glass tube. After incubation for 2-3 days at 4� C., free insulin is separated from antibody-bound insulin by charcoal separation. The assay sensitivity is 1-2 uU/mL. In order to measure the release of IRI into the cell culture medium of cells grown in tissue culture, one preferably incorporates radioactive label into proinsulin. Although any radioactive label capable of labeling a polypeptide can be used, it is preferable to use 3 H leucine in order to obtain labeled proinsulin.
To determine whether a GLP-1 analog has insulinotropic properties may also be determined by pancreatic infusion. The in situ isolated perfused rat pancreas assay is a modification of the method of Penhos, J. C., et al., Diabetes, 18:733-738 (1969). Fasted male Charles River strain albino rats, weighing 350-600 g, are anesthetized with an intraperitoneal injection of Amytal Sodium (Eli Lilly and Co.: 160 ng/kg). Renal, adrenal, gastric, and lower colonic blood vessels are ligated. The entire intestine is resected except for about four cm of duodenum and the descending colon and rectum. Therefore, only a small part of the intestine is perfused, minimizing possible interference by enteric substances with glucagon-like immunoreactivity. The perfusate is a modified Krebs-Ringer bicarbonate buffer with 4% dextran T70 and 0.2% bovine serum albumin (fraction V), and is bubbled with 95% O2 and 5% CO2. A nonpulsatile flow, 4-channel roller bearing pump (Buchler polystatic, Buchler Instruments Division, Nuclear-Chicago Corp.) is used, and a switch from one perfusate source to another is accomplished by switching a 3-way stopcock. The manner in which perfusion is performed, monitored, and analyzed follow the method of Weir, G. C., et al., J. Clin. Investigat. 54:1403-1412 (1974), which is hereby incorporated by reference.
The present invention also provides pharmaceutical compositions comprising a GLP-1 analog of the present invention in combination with a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art, and are administered individually or in combination with other therapeutic agents, preferably via parenteral routes. Especially preferred routes include intramuscular and subcutaneous administration.
Parenteral daily dosages, preferably a single, daily dose, are in the range from about 1 μg/kg to about 1,000 μg/kg of body weight, although lower or higher dosages may be administered. The required dosage will depend upon the severity of the condition of the patient and upon such criteria as the patient's height, weight, sex, age, and medical history.
In making the compositions of the present invention, the active ingredient, which comprises at least one protein of the present invention, is usually mixed with an excipient or diluted by an excipient. When an excipient is used as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the active ingredient.
In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active protein is substantially insoluble, it ordinarily is milled to particle size of less than about 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, mannitol, starches, gum acacia, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, sweetening agents or flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
The compositions are preferably formulated in a unit dosage form with each dosage normally containing from about 50 μg to about 100 mg, more usually from about 1 mg to about 10 mg of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient.
For the purpose of parenteral administration, compositions containing a protein of the present invention preferably are combined with distilled water and the pH is adjusted to about 6.0 to about 9.0.
Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to complex or absorb a compound of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
Another possible method to control the duration of action by controlled release preparations is to incorporate a protein of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.
Alternatively, instead of incorporating a compound into these polymeric particles, it is possible to entrap a compound of the present invention in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in macroemulsions. Such teachings are disclosed in Remington's Pharmaceutical Sciences (1980).
Similarly, the present invention provides a method for treating diabetes or hyperglycemia in a mammal, preferably a human, in need of such treatment comprising administering an effective amount of a GLP-1 analog or composition of the present invention, to such a mammal.
By way of illustration, the following examples are provided to help describe how to make and practice the various embodiments of the invention. These example are in no way meant to limit the scope of the invention.
Met-8 GLP-1(7-36)NH2 was produced by solid phase peptide chemistry on an Applied Biosystems (ABI) 460A peptide synthesizer using a MBHA resin (Applied Biosystems Inc., lot # A1A023, 0.77 mmol/g). All amino acids had their a-amino groups protected by the tert-butyloxycarbonyl (t-Boc) group. Those with reactive side chains had them protected as follows: Arg(Tos); Lys(Cl-Z); Trp(CHO); Glu(CHex); Tyr(Br-Z); Ser(Bzl); Asp(OBzl); Thr(Bzl).
The protected amino acids were activated in dichloromethane (DCM) with one half an equivalent of dicyclohexylcarbodiimide (DCC) per equivalent of amino acid to give the symmetric anhydride of the amino acid. However, arginine, glutamine, and glycine residues were activated by forming the 1-hydroxybenzotriazole (HOBt) esters of these amino acids (1:1:1 equivalents of amino acid, HOBt, and DCC in dimtethylformamide (DMF)).
Residues were sequentially connected from the C-terminal towards the N-terminal end with a series of coupling and deprotection cycles. A coupling cycle consisted of the activated amino acid undergoing nucleophilic substitution by the free primary amine of the previously coupled amino acid. Deprotection was the removal of the N-terminal blocking group Boc with anhydrous trifluoroacetic acid (TFA). This generated a free amine group after neutralization with diisopropylethylamine (DIEA).
The synthesis scale was 0.5 mmol. The concentration of functional sites on the MBHA-resin was 0.77 mmol/g; 649 mg of resin was used. A two fold molar excess of the symmetric anhydride was used for all of the amino acids. The C-terminal Arginine was coupled to the MBHA-resin via standard protocols. All residues were double-coupled. That is each residue was coupled to the resin twice. The second coupling was performed without a Boc deprotection step prior to re-addition of the amino acid. This helped to completely react all of the free amine groups on the resin. The tryptophan residue was quadruple coupled. After the second coupling step of each double-coupling cycle the N-terminal Boc croups were removed with anhydrous TFA and neutralized with DIEA.
The formyl side chain blocking group on the tryptophan residue was removed with piperidine in DMF prior to cleaving the peptide from the resin. After the peptidyl-resin was transferred to a 50 ml sintered glass funnel, it was washed several times with DCM and DMF. Then 3-5 ml of a 50/50 piperidine/DMF solution was added to the peptide-resin so that it was just covered. After 5 minutes the piperidine/ DMF was removed by vacuum and 3-5 ml of piperidine/DMF was added. After 10 minutes, the piperidine/DMF again was removed by vacuum filtration and 15-20 ml of piperidine/DMF was added. After 15 minutes the piperidine/DMF was removed and the peptidyl-resin washed with DMF several times followed by DCM. The peptidyl-resin was then placed into a vacuum oven (no heat) to complete solvent removal.
Once the amino acids were sequentially coupled, and the formyl group removed, the peptide was liberated from the resin by hydrolysis with liquid hydrofluoric acid (HF) at 0� C. for one hour using a Teflon reaction vessel. In the process of liberating the peptide, the C terminal hydroxide was displaced with an amide group from the MBHA-resin (see Matseuda and Stewart, Peptides 2:45 (1981)). For every gram of peptidyl-resin, 1 ml of m-cresol scavenger was added and 10 ml of liquid HF used. The scavenger prevented the reattachment of side chain blocking groups (released as carbocations) to the peptide. After one hour, the HF was removed by vacuum leaving a slurry of peptide, resin, and m-cresol.
The peptide was then precipitated in the HF reaction vessel with ice cold diethyl ether. The precipitate was transferred to a 150 ml sintered glass funnel along with several ether rinses. The peptide/resin physical mixture was washed several times with cold ether to remove residual HF and m-cresol. The second step was to extract the peptide away from the resin using 10% acetic acid in water (v/v) Vacuum filtration into a clean round bottom flask yielded a crude peptide solution.
The crude peptide solution obtained in Example 1 was run on reverse-phase analytical HPLC at pH 2.3. The chromatogram showed a major peak indicating that an appreciable amount of the desired product was formed and that preparative purification was warranted.
The entire crude peptide solution was run on preparative reverse phase HPLC at pH 2 under the following conditions:
Buffers: A=0.1% TFA, 10% acetonitrile B=0.1% TFA, 50% acetonitrile
Column: Vydac C18 (2.2�25 cm)
Temperature: approximately 20� C.
Detector: 280 nm
Flow: 2.0 ml/min
Gradient: 25% B to 100% B over 1,000 minutes
The title peptide eluted at approximately 34% acetonitrile as identified by analytical HPLC and electrospray mass spectroscopy. The approximate yield was 125 mg of 60% purity by analytical HPLC at pH 2.3.
This crude preparation was then run on preparative reverse phase HPLC at pH 7.7 under the following conditions:
Buffers: A=0.1M (NH4)HCO3, 10% acetonitrile B=A, 50% acetonitrile
Column: Vydac C18 (2.2�25 cm)
Temperature: approximately 20� C.
Detector: 280 nm
Flow: 2.0 ml/min
Gradient: 35% B to 70% B over 1,000 minutes
The title peptide eluted between 32% and 37% acetonitrile as identified by analytical HPLC. The approximate yield was 10%.
Thr-8 GLP-1(7-37)OH was prepared in substantial accordance with Examples 1 and 2, except that the peptide was released from the solid phase such that the C-terminal hydroxyl group remained.
a) Rat, GLP-1 receptor, membrane preparation:
The published DNA sequence for the rat GLP-1 receptor (Thorens B., et. al. Proc. Natl. Acad. Sci. U.S.A. 89:8641-8645 (1992) and the dihydrofolate reductase resistance marker gene were used in conjunction with PCR techniques to construct an expression vector. The DXB-11 variant of the chinese hamster ovary (CHO) cell line was transformed with the vector, resulting in a recombinant CHO cell line that expressed the rat GLP-1 membrane receptor.
Cells were grown and harvested, and a membrane preparation was obtained by first washing the cells with PBS buffer, then twice washing with cold buffer (25 mM HEPES, 2 mM MgCl2, 1L mM EDTA, 20 μg/ml Leupeptin, 1 mM PMSF, 2μg/ml Aprotinin, 50 μg/ml Trypsin Inhibitor, pH 8.0) and resuspending in buffer. The cell suspension was lysed in a glass Teflon homogenizer, and the resulting sample was then centrifuged at 35,300� g for 30 minutes at 4� C. The supernatant was removed, and the pellet was resuspended in cold buffer and homogenized. Aliquots were stored at -80� C.
b) Cyclic AMP (cAMP) Assay:
A sample of the membrane preparation was pre- incubated with a test compound or a control compound in buffer (25 mM HEPES, 0.2% (w/v) BSA, pH 7.6) at 32� C. for 10 minutes. Reaction buffer (final concentration: 25 mM HEPES, 0.2% (w/v) BSA, 2.6 mM Mg, 0.8 mM ATP, 0.1 mM GTP, 5 mM creatine phosphate, creatine kinase 50 U/ml, 0.2 mM IBMX, pH 7.6) was added and incubated for an additional 30 minutes. Incubations were stopped by adding 10 mM EDTA.
Production of cAMP was assayed using a fluorescent tracer-immuno assay method. In brief, after the incubation was stopped, fluorescent tracer (cAMP-b phycoerythrin conjugate) was added followed by the addition of affinity purified anti-cAMP rabbit antiserum. After incubation at room temperature for 45 minutes, anti-rabbit IgG coated assay beads were added and incubated for an additional 15 minutes. Plates were then evacuated and read on a Pandex PFCIA reader.
In this assay, a known insulinotropic agent such as GLP-1(7-37)OH showed decreasing fluorescent intensity due to increased cAMP concentration. Fluorescent intensity values were correlated to rate of cAMP production (pmol/min/mg). Conversely, agents having no insulinotropic action failed to stimulate production of cAMP and therefore showed no decrease in fluorescent intensity. Percent receptor affinities are shown below:
______________________________________GLP-1(7-37)OH 100% Met-8 GLP-1(7-36)NH2 16.6% � 5.8% Thr-8 GLP-1(7-37)OH 2.8% � 0.7%______________________________________
__________________________________________________________________________# SEQUENCE LISTING - - - - <160> NUMBER OF SEQ ID NOS: 2 - - <210> SEQ ID NO 1 <211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Xaa at position 1 is - #chosen from His, D-histidine, desamino-histidine, 2-amino-h - #istidine, beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine. <220> FEATURE: <223> OTHER INFORMATION: Xaa at position 2 is - #chosen from Met, Asp,Lys, Thr, Leu, Asn, Gln, Phe, Val a - #nd Tyr. <220> FEATURE: <223> OTHER INFORMATION: Xaa at position 15 is - #chosen from Glu, Gln, Ala, Thr, Ser, and Gly. <220> FEATURE: <223> OTHER INFORMATION: Xaa at position 21 is - #chosen from Glu, Gln, Ala, Thr, Ser and Gly. <220> FEATURE: <223> OTHER INFORMATION: Arg at position 30 is - #amidated, if Gly at position 31 is absent. <220> FEATURE: <223> OTHER INFORMATION: Gly at position 31 may - # be absent. Ifabsent, Arg at position 30 is amidated. <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic construct - - <400> SEQUENCE: 1 - - Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Val Se - #r Ser Tyr Leu Xaa Gly 1 5 - # 10 - # 15 - - Gln Ala Ala Lys Xaa Phe Ile Ala Trp Leu Va - #l Lys Gly Arg Gly 20 - # 25 - # 30 - - - - <210> SEQ ID NO 2 <211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Xaa at position 1 is - #chosen from His, D-histidine, desamino-histidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, - #and alpha-methyl-histidine. <220> FEATURE: <223> OTHER INFORMATION: Xaa at position 15 is - #chosen from Glu, Gln, Ala, Thr, Ser, and Gly. <220> FEATURE: <223> OTHER INFORMATION: Xaa at position 21 is - #chosen from Glu, Gln, Ala, Thr, Ser, Gly. <220> FEATURE: <223> OTHER INFORMATION: Arg at position 30 is - #amidated, if Gly at position 31 is absent. <220> FEATURE: <223> OTHER INFORMATION: Gly at position 31 may - # be absent. Ifabsent, Arg at position 30 is amidated. <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial - #Sequence:Synthetic construct - - <400> SEQUENCE: 2 - - Xaa Met Glu Gly Thr Phe Thr Ser Asp Val Se - #r Ser Tyr Leu Xaa Gly 1 5 - # 10 - # 15 - - Gln Ala Ala Lys Xaa Phe Ile Ala Trp Leu Va - #l Lys Gly Arg Gly 20 - # 25 - # 30__________________________________________________________________________
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|International Classification
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|European Classification
|Jun 21, 1999
Owner name: ELI LILLY AND COMPANY, INDIANA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HOFFMANN, JAMES ARTHUR;REEL/FRAME:010042/0410
Effective date: 19980227
|Mar 27, 2001
|Certificate of correction
|Mar 31, 2003
|Fee payment
Year of fee payment: 4
|May 28, 2003
|Maintenance fee reminder mailed
|May 30, 2007
|Maintenance fee reminder mailed
|Nov 9, 2007
|Lapse for failure to pay maintenance fees
|Jan 1, 2008
|Expired due to failure to pay maintenance fee
Effective date: 20071109


text:
Live with community and luxury
When choosing a place to live there are many factors to consider. Avalon Apartments, off Milwaukee Avenue and 34th Street, is the latest luxury apartment complex in Lubbock that has everything you need in your residence as well as amenities you didn't know you wanted. Along with the convenient location just minutes from West End Shopping Center — Lubbock's fast-growing area full of the latest restaurants and shopping — Avalon offers the highest of luxuries in a modern rustic style.
Avalon's brand new apartments are designed with an open, welcoming feel to make your apartment feel less like a rental and more like home. Inside you will find a large, open kitchen with an island that looks into the living room along with wide doors to help open up the design. New stainless steel appliances give you a modern, upscale feel for your new home and granite counter tops compliment the rustic feel in the kitchen and bathrooms.
Catalina is the design name for Avalon's 800 sq. ft. one-bedroom, one-bath apartment that takes its name from Santa Catalina Island off the coast of California. This island is home to breathtaking views, zip-lining, beach houses and classy restaurants. The "hub city" on the island is Avalon. Avalon Apartments strives to bring the views and resort-feel of Avalon back to Lubbock. The white rock of the apartments and clubhouse symbolize the white sand beaches, bringing the outdoors inside your home; the beautiful year-round heated pool brings the warm waters off the coast to your community.
Everything about Avalon Apartments aims to make you comfortable and safe, such as the fire tables and lounge areas outside the clubhouse, the covered gas grilling stations available for use anytime, the courtyard entrances and keyless entry to your apartment through your smartphone's Bluetooth. All these amenities and more provide the feeling of a custom home more than an apartment.
"Community is really important wherever you live," says Sara Fierro, property manager of Avalon Apartments. Avalon hosts community events like yoga sessions, barbecues, annual wine tastings and more to help you socialize and connect with your neighbors. The clubhouse is available to socialize too, with an activity room for watching the game or hosting parties. The clubhouse also has a gym accessible 24 hours a day for whenever you feel the urge to exercise. Avalon's resort-style heated pool sits outside the clubhouse too, along with sun decks and tanning ledges. A private park is being built that will only be available to Avalon residences — something completely new to Lubbock. The park will be complete with a jogging track, fire tables, gas grilling stations and plenty of space to relax and read a book or spend time as a family. The park will be lit to add safety at night. All these features add to your living experience and extend your apartment into the outdoors.
"We want people to feel heard and cared about. That's what really makes Avalon feel like home," says Sara. Whatever your need or concern, talk to the staff at Avalon. If you'd like to see a model, visit Avalon Apartments at 3420 Milwaukee Ave. or call (806) 793-2020. Visit AvalonLubbock.com to see more.
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text:
New Zealand: Kaikôura Harbour restoration progressing wellNews // February 7, 2017
Scoop reports that restoration of Kaikōura Harbour after last year's magnitude 7.8 earthquake is expected to be completed by the middle of the year.
Civil Defence Minister Gerry Brownlee has announced progress on the slipway, ramp and channel deepening as a result of the November 14 earthquake lifting the seabed between one and two metres in some areas.
"At the moment access is severely restricted and the harbour channel is so shallow, it can only be used four hours a day – two hours either side of the tide," Mr Brownlee says.
"Commercial operators can only schedule between 20 per cent and 40 per cent of their previously planned tours and the district's commercial fishing industry has also been adversely affected.
"The importance of tourism and fishing cannot be underestimated – the annual tourism spend in Kaikōura is NZ$120 million (domestic and international) and the value of the annual fishing catch is approximately NZ$25-30 million.
"The NZ$5 million government grant meant work could start straight away on repairing and upgrading this essential piece of infrastructure."
The full story is at:
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Posts Categorized: Campus Program
Star Volunteer – 4 Paws UK – August/November 2014
4 Paws University Involvement
How to Get Started 4 Paws for Ability University Program accepts new program participants twice a year, fall and spring semesters. Summer session is reserved only for returning handlers, new participants are unable to begin in the summer. If you are attending classes at one of our 13 official campus programs, you should contact them for… Read more »
Puppy Raisers 2012
Meet a Puppy Raiser Tori Shishler | University of Kentucky Foster, Fall 2012 I have people come up to me all the time and ask me how I can give up the dog after getting attached to him and that they could never do the program because of that. I know that its going to one of… Read more »
Puppy Raiser – Kaitlyn Schmerge
Meet a Puppy Raiser Name: Kaitlyn Schmerge School: Sinclair Community College Major: Veterinary Technology Dogs fostered: Fostered 5 dogs(4 long term fosters and one short term foster) – Cinderella, Cragen, BelleStarr, Gibbs, Deeks To fully put into words what 4 Paws for Ability has done in my life is nearly impossible. Starting college, I didn't know where it… Read more »
Puppy Raiser – Melanie Repella
Meet a Puppy Raiser Name: Melanie RepellaSchool: The Ohio State UniversityCampus Program Role: PresidentDogs fostered: I've fostered six dogs throughout my time with 4 Paws. Atlantis. Amar. Shrek. Raguel. Gally. Saul. Each dog has left with a piece of my heart and knowing that they belong with someone who needs them more than I do… Read more »
Puppy Raiser – Amanda Prater
Meet a Puppy Raiser Name: Amanda Prater School/Degree: University of Kentucky, B.A. Psychology School/Degree: University of Dayton, School Psychology Graduate ProgramDogs fostered: 8 My experience as a college foster for 4 Paws for Ability has been invaluable. I began fostering for 4 Paws during my undergraduate career as a junior at UK. While at UK, I fostered 4… Read more »
Puppy Raiser – Heidi Vollrath
Meet a Puppy Raiser Name: Heidi Vollrath School: University of Kentucky Major: Business Management Dogs fostered: 8 Dogs The impact 4 Paws has had on my life is difficult to put into words. I can truly say I would not be the person I am today without 4 Paws for Ability at the University of Kentucky. What started… Read more »
Puppy Raiser – Gabrielle Bowers
Meet a Puppy Raiser Name: Gabrielle BowersSchool: University of Kentucky// Traditional FosterDegree: BA in Learning & Behavior Disorders in Dec 2014, Masters in Behavioral Psychology from Ball State in May 2016.Dogs fostered: I fostered 5 dogs through the UK program & 2 through the traditional fostering program, bringing my total to 7 dogs. The university… Read more »
Puppy Raiser – Bennett Baber
Meet a Puppy Raiser Name: Bennett BaberSchool: University of KentuckyDegree: BA in Health Communication, May 2015Dogs fostered: I fostered 6 dogs through the university program and 3 dogs as a traditional foster for a grand total of 9! The university program benefited me in more ways than I can count. Not only did it help… Read more »


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Added to Shopping Bag
Loafers Preppy Black Formal Shoes
Listed by Ginger Closet
Size: 5
Price: $44.00
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Loafers Preppy Black Formal Shoes
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- Exterior scuffs or marks
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Selling a gently used Scott David black Loafers. Size 5. Made in Spain. Please note additional picture is an idea of how you may style the loafers but does not reflect the exact item.
Price includesTradesy Safe Shipping.
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Mon 5/1/17 - Thu 5/4/17 (5-8 days)
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You can see the panic in Davido's face when this snake (an albino python) woke up on his body. It was all fun and games when the snake was asleep but once it started moving, it was a whole different story.
Davido had one of these snake charmer guys put a snake that was asleep over his body to pose for photos. Once the snake woke up tho… The game changed.
Watch the video here:
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Get All Latest Android APK Games And Apps Here
Get All Latest Android APK Games And Apps Here


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Exercise Mats For Treadmill - Chinaprice: contact company for price
Material: PVC Foam
Features: good toughness
Anti- skid
Lower noise
Non- Toxic
Use for: perfect for steppers, exercise bikes, step- aerobics, or just stretching. Protect floor' s surface from scratches dings and normal wear and tear caused by machines.
LOGO print: YES
Easy clean
Packing: 1pc/ shrink film, 6pcs/ ctn. or by buyer
Company Contact:
- Posted By: Suzhou Globelink Imp&Exp Co. Ltd
- Phone: 086-512-62535711
- Address: No.368 Suya Road, Suzhou, China
- Email:
- Website: http://www.szglobelink.com/
Published date: May 22, 2014 -
- Business Description: The company is situated in East of Jiangsu province, close to Shanghai,
having long term close cooperation with many leading domestic production
factories, we can make rapid reaction to any requests from the
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Earth Pledge
"I pledge allegiance to the Earth, and all the life it
supports. One planet, in our care, irreplaceable, with
respect and sustenance for all"
More Info
Photo Gallery
The Story
taken at Burning Man 2007
Photo compilation -
thanks to Janabanana and Dusty for a lot of these photos
Maia's photos
Bellequa's photos
Blaze's photos
Lisa kicks butt in the
Thunderdome - video
taken at Burning Man 2009
Mr. God's Photos
Below are photos of the
process of building the Amphibia structure
The planet named Amphibia
is wet and deeply green.
It's quite a bit like Earth was once,
when dinosaurs were seen.
And on this planet far away
a special frog was born one day…
Infinity!—a hero's name,
this frog was destined
for Earth fame.
They told her the assignment—to save the frogs on Earth.
"Our cousins are endangered there,
the humans rule without a
They're stuck on greed and foolish pride, they do not see
that deep inside,
They're part of nature, not above it,
they need to learn to
truly love it.
Act with LOVE and off you go—
be sure to give them all a show!"
And off she flew through
deepest space
toward a new and different place.
She flew too fast for us to see,
She earned her name—Infinity!
At last she came to planet Earth,
a precious thing of unknown worth.
Where to start?
She made her plan
to visit first the Burning Man.
The Amphibia story is excerpted and altered
from an upcoming children's book by Juanita