Will this ever end? @31 months

I don’t post very much at all. I come in here to try to get support. I feel like I’m actually losing my mind. I am 31 months in and the last 3 months have been some of the worst. I’ve never felt more alone, scared , desperate and sorrowful in my whole life. Everyday dealing with this intense pain, neurological shut down and anxiety is just overwhelming. I honestly never knew the things that are happening to my body could happen to anyone. At least not all at one time to a body. It saddens me to not see very many recovery stories. I feel like if I ever come out of this it will be a miracle. I feel like I actually died in many ways a couple years ago. I really just feel like I don’t have what it takes anymore to go on. I haven’t taken one medication through these almost 3 years. I’m too scared to. At this point I feel like it doesn’t matter. I’m actually afraid of dying from this and my son not having a mom. I’m a single mom so I don’t know what would happen to him. I know this is dark..I’m really not a dark person. I just needed to vent to the only people that I know understand. Thank you

Anxiety meds

Hi only problem is severe anxiety brain fog nothing physical Iv went over two years now med free on other peoples advice but now I’m giving in to meds it’s going on to long I can’t do it anymore Can anybody please recommend anxiety meds that have worked for them thanks

So scared!

Hi all!

This is my first post, although I’ve been to visit the site several times in the last 6 weeks. It’s helped (and scared) me in my darkest moments. I received 36 units of botox on Feb. 6th in my forehead and glabella and have experienced nothing but pure terror and pain ever since. As soon as the 3rd day after, I was having crazy panic attacks out of nowhere (something that never happened before), inability to eat, nausea, vertigo, insomnia, head pressure, etc. As many of you, I’ve went to the ER the second week after because it got to the point where I knew something was seriously wrong, but was sent on my way with Ativan since all tests came back normal. “Must be the anxiety of not being able to raise your forehead,” the doctor said.

Been to my general practitioner who gave me Zofran for nausea. He also did blood work since I had a fever of 101 degrees. Everything came back fine so he said it must be a virus or auto immune. I told him I thought it was the botox and he said it’s out of my system by now and doesn’t cause these symptoms. I got in with a neurologist next who said what I was experiencing was definitely from the botox (finally!) and unfortunately instigates these symptoms in people who are susceptible, especially with injections to the face. He diagnosed me with migraine with aura brought on by the botox injections  in individuals with type IV hypersensitivity. He said my symptoms would improve in 6-8 weeks. Well it’s been 6 weeks and I still battling with symptoms every day. I will say that I stopped getting nauseous after every meal at the 4 week mark, but that’s the only thing that’s improved. According to this site, there are people who are months into this with little relief. That scares me so much. I cry every day.

I take 400 MG of Motrin 3x’s a day, which is the only thing that’s really helped my symptoms. I drink a gallon of water every day as well. I have to take Lorazepam at night to sleep. If I don’t, it’s a sleepless night, which makes my symptoms worse. My neurologist wants to put me on Nortriptyline, but after my first day taking it, I felt so high. Didn’t like it at all so I stopped hoping that this mess would end soon. I don’t know if I can last much longer in this state though. I know I’m one of hundreds saying the same thing on this site, but I guess I’m looking for any kind of encouragement I can find.

 

 

Potassium

I know people here use magnesium a lot and I did too in the beginning. A *lot* of magnesium, especially on my neck since the muscles there were so weak, and twitching. I was also drinking a lot of coffee since it is a source of magnesium and also an antioxidant. It is very hot here, and my autonomous nervous system is flipping out, but also my nerves are trying to heal generally and that means the uptake of lots of b vitamins for cell repair. Plus I was *taking* b vitamins, a crapton of them.

Also, I was given prednisone and antibiotics, and drinking lots of fluids. At a certain point I just kept peeing and peeing and I was absolutely *parched*. My eyelids were fluttering like I had become electrified.

It was horribly, horribly frightening.

So last week I ended up in the ER, with a fever, shaking like mad. I couldn’t sit down because I would start to shake, sitting — the only position I was not trembling like a leaf in was standing up. I would try to sit on my couch during the day or lie down — shaking, shaking, shaking. So exhausted I was edging up onto psychotically terrified.

So in the ER they did the whole full thing. EKG, blood, urine, liver enzymes, blood cultures for bacteremia, septicemia — everything.

There was nothing wrong.

Except for one thing.

My potassium level was 2.5, which is the borderline cutoff for getting an IV bag of potassium. A cellular emergency. B vitamins and nerve and cell repair all use potassium channels to get nutrients into your cells. If there is cell damage, there is usually an intracellular shift that moves potassium out of the serum and into the cells — add that to stress, dehydration, antibiotics, a neurogenic bladder, low grade fever and diarrhea; and steroids and there is the potential for an actual *emergency* loss of potassium.

Just keep it in mind. You might need magnesium too but especially if you can’t eat much ask your doctor to pull a K ( potassium) value. I’ve read a couple of stories on here that describe shaking, dry mouth, extremely frequent urination and muscle weakness — all these things could be some other disease process but they could *also* be potassium loss. Potassium usually stays very stable in serum *unless* something really bad or strange has happened. And…something bad and strange *has* happened.

Also partial paralysis can be caused by low potassium, like if your hands dont seem to be working right, mouth twitches, tics, etc. It’s not because the muscles can’t contract, it’s because they can’t *release*.

When the muscles aren’t working correctly there are a couple of possibilities as to why. One is nerve damage or derangement — obviously possible with botox poisoning victims. However all the salts are involved as well. If you are losing salt — chloride, magnesium, potassium and calcium — these are things that make the body run when they’re in balance; they’re the conduit that enables the electrical charge that fires all muscle, nerve and brain impulses. If you are low on any of them, the effect will be terrifying and outrageously weird.

I am taking nearly illegal levels of potassium at the moment. US RDA for potassium is something like 3300 milligrams and one regular potassium pill from the drugstore is 99 mg — I’m taking about 30 of these a day ( or I was, I am cutting back now) and resting in a very cool room and staying hydrated. I’ve been doing that for the past four days. Last night was the first night in I don’t know how many months that I did not feel like I had an electrical current running through me all night long. It’s still not completely resolved and it still freaks me out horribly but it is a lot better for now.

I’m worried about my eyes.

When I got botulism I had terrible blurred and double vision that hasn’t really resolved six months later. I was glad when I could use the left side of my face again and that seemed to help with eye movement. I thought it was just the paralysis, and was waiting patiently for it to resolve.

It is better, somewhat. The strabismus is barely noticeable. No nystagmus. I still can’t focus close up; they call that “lack of accomodation” of the eye and it has to do with the ocular nerve. Just waiting patiently some more for that to resolve I guess, if it ever does.

Actually the reason I am hopeful about that is that I read somewhere the “loss of accommodation” is the very last thing to resolve in a botulism poisoning.

But I have soooo many eye floaters and weird cobwebs in my vision. They worry me. It’s like there’s some kind of screen in front of my retinas all the time. I haven’t seen “flashes of light” or red spots so I don’t think it’s my retina detaching, especially since it’s gone on so long. But the floaters are horribly distracting and worrying.

Anybody else have this, and have it resolve?

Saunas?

Anyone a regular user of saunas and do they help?  Read this article about detoxification:  http://fitness.mercola.com/sites/fitness/archive/2015/03/13/benefits-using-sauna.aspx?e_cid=20150322Z1_SNL_NB_TFA_1&utm_source=snl&utm_medium=email&utm_content=tfa1&utm_campaign=20150322Z1_SNL_NB&et_cid=DM70510&et_rid=885274009

 

MRI with contrast

I think I remember reading in the past that some of us have had MRIs with contrast? My doctor has ordered one for problems with my leg and I’m nervous about the contrast. If you’ve had one, can you post if you had any reaction or problems with the dye?

Thanks everyone,
j

Migraines with aura

I attempted to go back to work today after a years sick leave and spent a couple of hours deleting the 6000 odd emails ive accumulated. After a few hours I started to get tired and a little fuzzy and then bang I got hit with a migraine with aura. Both eyes were effected for several hours and then my vision returned and I was left with headache and feeling a little nauseous. I wanted to post this because I developed migraines 12 to 18 months after my botox poisioning and I’ve continued to get them for 7 years now. They seem to coincide with my period and also long periods on the computer and under fluorescent lighting. Id never had a migraine before botox. Does anyone have any remedies they can suggest?

Enough!

I’m a 53 year old very athletic woman, I play tennis, walk anywhere from 6-7 miles approx. 6 days a week and hit the fitness center on occasion.  And I am sick by what I believe Botox has done to me both mentally and physically.   Finding this site has been my salvation, it has helped to keep me positive and fighting through this miserable experience.

I have had approx. 4 or 5 injections, my last on being on April 3, 2014, 54 units in my forehead and around my eyes.  Three weeks later it all began to fall apart.  I started having some very concerning health issues, it began with having trouble breathing one day.  I tried to stay calm, thinking it must be some sort of indigestion, well low and behold I did burb,  but it was as if it had to fight it’s way out.  Then one day I noticed that my tongue was quivering and fasciculating, along with all of the other muscles in my body, and I mean pounding.  My muscles ached like never before, I would be sitting on the couch and be in pain, without even moving,  then I would get this spasm in my left calf that was terribly painful.  I’ve been athletic all my life, I’ve never had a muscle spasm!.  So then I start having trouble speaking, I was on the tennis court, just trying to say “good shot” and it was like I had to fight to get the words out of my mouth, thank heaven, that only lasted a few weeks.  When I would go to work out I would come home literally with my muscles trembling,  when I would try to do a plank my arms would shake like jello, when I was walking I was afraid I was going to fall because my muscles weren’t supporting me.  My arm would just jerk, for no reason, out of the blue.  Then the real fun set in, trouble swallowing, anxiety and depression, I would wake every day with just this overwhelming feeling of dread.

The trouble swallowing and my tongue quivering led me to begin with a visit to an ENT.  I was diagnosed with laryngeal and pharyngeal dystonia, which is caused by a neurological problem which they treat with….Botox!  I could tell by the Dr.’s face I was in trouble.  My voice was also very hoarse.  Wants me to see a neurologist immediately.

This is when I began to utterly fall to pieces.  I was sure it was either ALS or Parksinson’s, Google really can a times be one of the worst inventions ever.  I contacted my GP and asked for a prescription for Xanax, the anxiety I was experiencing was overwhelming.

So begin the visits to two neurologist, who conducted muscle and nerve conduction tests, I had blood work, two MRIs and various visits to my GP.  Guess what?  Came up with nothing.   Am I crazy???

In regard to the trouble swallowing I noticed that if I had a cocktail I could swallow.  What the heck?   I couldn’t each much during the day, so at night I have a cocktail and eat like a truck driver.  Well, then put two and two together and found that the Xanax helped with the swallowing a bit as well, but not as much as the alcohol.  I was grateful for that because I couldn’t go around drinking all day and I was freakin hungry.  The ENT said it was because it was relaxing the muscle.

One day I had the TV on and heard a commercial for Botox and heard that it can cause trouble swallowing!  I’m embarrassed to admit that I did not due any due diligence regarding the strongest neurotoxin on earth that I was injecting into my body!

Months pass and I notice that my tongue is not quivering anymore and my voice isn’t as hoarse.  I went back to the ENT and asked him to bear with me and hear me out, I think this has been caused by Botox.  Now the man works with it to treat the condition I was diagnosed with so I was expecting him to disagree with me.  He scoped my throat and said he saw improvement!  He said, “I think you figured this out for yourself”.  He agreed with me!!  I almost jumped out of the chair with sheer joy!  He also told me the problem swallowing could last anywhere from 6 months to one year!

After that I started to calm a bit, still dealing with all of the symptoms, reading this site to stay strong,  but hoping we’re right and just trying to cope through it.  Then I get a call the other day from my GP and he asked if I’m buying into this.  Oh no, please, I can’t go there again.  He wants me to go back to the ENT for a recheck.  I went in, making sure not to take any Xanax so I would get a clear result. He said the dystonia was 90% better but he wants me to stop the Xanax in the morning to make sure it’s not masking something and I truly am improving.  Enough, please!  Now I’m a wreck again.  What if I have been masking something?  I can’t do this again.

I’m going to try to stay positive, I’m pretty darn sure I’m heading in the right direction!  I’m not having trouble speaking anymore, my muscles don’t hurt, albeit they do still fasciculate, but not as hard, I’m not cramping anymore and the depression has lifted.  I am still struggling a bit with anxiety, but having trouble swallowing for close to a year gets old.

My questions for any of you are:  Has anyone had the same diagnosis of dystonia and has anyone had trouble swallowing for such a long time.

My helpful hints are fish oil and especially magnesium.  Not all magnesium is the same, some are not absorbed as well.   I’m using Magnesium Taurate and truthfully find it amazing in calming my muscles.  Also a cocktail for swallowing….I’m probably going to wind up in AA, I think I’ll send the bill to Allergan, or whoever just bought them out.

Thanks for reading my ridiculously long dissertation.   Any and all support, suggestions, ideas, anything would be enormously appreciated.  And for those of you who began this site, I thank you from the bottom of my heart.  For those of you suffering I wish you strength!

 

I

 

 

 

 

Sound Therapy: Meditation. Quiet. Pleasing.

I saw a Julian Treasure talk on TED TALKS and it held special relevance for the nerve damage from botulism. Sound intolerance seems to be a common thread in botulism poisoning but again, it is subjective and hard to measure. But in this video Julian explains the assault from sounds and physiological reactions we have. He also offers recommendations to combat the assault and create a better environment.

Has anyone noticed a sound intolerance (includes chaos, visual clutter, and children intolerance) and tried this strategies?

The Shaking.

So I am wondering how many people here had this shaking. It is torturous. I have no idea when the waking/standing/sitting tremors are coming but in public they distract me because I am trying to stay present while my body is just trembling, all the time. Or actually not all the time, sometimes it surprises me and stays quiet. Sometimes for hours. And I’ll think, oh, maybe it’s stopping, maybe it will stop forever. This happens maybe 100 times a day.

I can be fine for a while, to the point I almost forget, and then I’ll reach for my phone and try to type a text and my thumb will be wavering, my eyes will go blurry for a little bit. And then later…somehow, it stops again.

I have tried everything to stop the buzzing at night. The back of my head, my back, upper and lower, my legs. I have drugs — flexeril seems to work and then it doesn’t, valium, then it doesn’t, magnesium, sometimes, potassium, sometimes.

The only other time I’ve ever experienced something like this was when I had a high fever and I was very ill. Only I am not ill. My heart is fine, my blood is fine, I got an EKG and so on. There is nothing that emergency medicine has pinpointed that will kill me. There is nothing that the neurologist can help with. My CK values are normal, no muscle wasting.

I gave up all caffeine — maybe I was intolerant suddenly. No. My diet has changed a thousand times. Nothing works — and beyond that everything I try I try hopefully, waiting for it to resolve, and every once in a while it will seem like something is working, and it turns out, that’s not it.

The past two days I now have this twitch in my right eyelid. I tried putting a little topical magnesium on it, it went away for a couple hours and woke up buzzing through my back and neck, and this little pull in the muscle over my eye.

While somehow my heart beats fine and my lungs work and my stomach digests food and for the most part I can think. Actually it’s not true that I am digesting food – strange things happen, whenever things start moving in my intestines I’ll feel faint and sort of awful for an hour or two. Then it will stop.

The thing is if it just stayed like this — if someone said well you’re just going to have to live with this for the rest of your life, maybe I could adjust. I would have to. But I keep worrying it’s going to get worse and I’m just going to get less and less control over my body.

I’m alive, but I can’t function. I’m too tired and upset to even worry about the future or what is going to happen next. The only thing that I’m worried about is if or when this will ever stop, and whether it will get worse.

I am starting to think it is permanent, and like I said…well, people have afflictions. But if it gets worse…

Botox at Real Self.

Just want to mention that I’ve tried to post a review of “my botox experience” over there and also make a forum comment about it.

Not only did it not get posted, there wasn’t even a response saying it was rejected. Just…delete! And crickets.

Also as an aside, a lot of what I have now read on there from these plastic surgeon experts is plain wrong. Just plain wrong. Not wrong because I am ill and angry, but wrong *factually*. I had to stop reading it because people like me are not allowed to respond.

Did anyone here contract classical botulism?

I am wondering this because I read people saying that they had terrible symptoms, but not really the published common symptoms of botulism.

I had it. I had facial descending paralysis, blurred and double vision, tight throat, arm weakness, a paralyzed left shoulder and back, and I am quite certain I had paralytic ileus. It doesn’t sound *so* dramatic when you write it down but it was and is really brutal.

I am really confused about what is in the literature. It seems like they’re all saying that people who contract classical botulism make a “full recovery”but I feel like I am getting sicker. Certain things are resolving – like I can use the left side of my head again, and I think my neck is not as weak.

But otherwise I am really really ill. In fact when I compare how I felt generally a month ago to how I feel now it’s starting to really scare me. I am so weak. And foggy. My stomach doesn’t work. I’ve been running a fever and having to pee almost every five seconds. I’m intensely dehydrated. I am starting to obsess over what happens on the toilet since it’s usually really concerning.

It’s like the flu that never leaves.

And I am starting to worry about whether I will continue to get worse. Time is supposed to heal but certain things are *not* gone, and they should be. Like the vertigo and the fact that I can’t use my hands very well. I shake and want to lie down after about two hours of regular activity after I wake up.

I can *not* take the heat. It’s like my autonomous nervous system just can’t hack any change in temperature. I am clumsy because my left side does not work and my proprioception is way off. I feel stiff and my throat will tighten up.

I have no energy to do anything. In the middle of the day I’ll go bright red and start to sweat and feel feverish, that will last until about 10 PM and then I’ll feel “normal”for a while.

A little while.

I am trying to pack to move out of here and just making the boxes and taping them together is a big deal. I feel like I don’t know my body. I can barely stay on one train of thought.

You all got better eventually right? This is just a phase…right?

this should come as no surprise

http://www.businessinsider.com/drug-trial-reports-2015-3

“Scientific studies that show something doesn’t work… are much less likely to get published.

Drug companies may keep such data quiet, or scientific journals may be unwilling to accept papers with negative results because they’re not as exciting. Over time, this pattern creates the impression that certain medicines are more effective than they really are, because there are no papers out there saying otherwise.”

(( Read more: http://www.psmag.com/health-and-behavior/clinical-trials-no-reporting#ixzz3UPtYeJ8t ))

http://www.businessinsider.com/fda-inspections-reveal-fraud-2015-2

“The FDA has repeatedly hidden evidence of scientific fraud not just from the public, but also from its most trusted scientific advisers, even as they were deciding whether or not a new drug should be allowed on the market. Even a congressional panel investigating a case of fraud regarding a dangerous drug couldn’t get forthright answers. For an agency devoted to protecting the public from bogus medical science, the FDA seems to be spending an awful lot of effort protecting the perpetrators of bogus science from the public.”

(( Read more: http://www.slate.com/articles/health_and_science/science/2015/02/fda_inspections_fraud_fabrication_and_scientific_misconduct_are_hidden_from.html#ixzz3UPskcUuS ))

Orthostatic Hypotension from Botox

:). I actually just put the “from botox” in the subject for the search engines. This is really for Twix12:

Abstract

Orthostatic hypotension is a chronic, debilitating illness that is difficult to treat. The therapeutic goal is to improve postural symptoms, standing time, and function rather than to achieve upright normotension, which can lead to supine hypertension. Drug therapy alone is never adequate. Because orthostatic stress varies with circumstances during the day, a patient-oriented approach that emphasizes education and nonpharmacologic strategies is critical. We provide easy-to-remember management recommendations, using a combination of drug and non-drug treatments that have proven efficacious.

Orthostatic hypotension is a chronic, debilitating illness associated with common neurologic conditions (eg, diabetic neuropathy, Parkinson disease). It is common in the elderly, especially in those who are institutionalized and are using multiple medications.

Treatment can be challenging, especially if the problem is neurogenic. This condition has no cure, symptoms vary in different circumstances, treatment is nonspecific, and aggressive treatment can lead to marked supine hypertension.

This review focuses on the prevention and treatment of neurogenic causes of orthostatic hypotension. We emphasize a simple but effective patient-oriented approach to management, using a combination of nonpharmacologic strategies and drugs clinically proven to be efficacious. The recommendations and their rationale are organized in a practical and easy-to-remember format for both physicians and patients.
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WHAT HAPPENS WHEN WE STAND UP?

When we stand up, the blood goes down from the chest to the distensible venous capacitance system below the diaphragm. This fluid shift produces a decrease in venous return, ventricular filling, cardiac output, and blood pressure.1

This gravity-induced drop in blood pressure, detected by arterial baroreceptors in the aortic arch and carotid sinus, triggers a compensatory reflex tachycardia and vasoconstriction that restores normotension in the upright position. This compensatory mechanism is termed a baroreflex; it is mediated by afferent and efferent autonomic peripheral nerves and is integrated in autonomic centers in the brainstem.2

Orthostatic hypotension is the result of baroreflex failure (autonomic failure), end-organ dysfunction, or volume depletion. Injury to any limb of the baroreflex causes neurogenic orthostatic hypo tension, although with afferent lesions alone, the hypotension tends to be modest and accompanied by wide fluctuations in blood pressure, including severe hypertension. Drugs can produce orthostatic hypotension by interfering with the autonomic pathways or their target end-organs or by affecting intravascular volume. Brain hypoperfusion, resulting from orthostatic hypotension from any cause, can lead to symptoms of orthostatic intolerance (eg, lightheadedness) and falls, and if the hypotension is severe, to syncope.
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A DECREASE OF 20 MM HG SYSTOLIC OR 10 MM HG DIASTOLIC

The consensus definition of orthostatic hypotension is a reduction of systolic blood pressure of at least 20 mm Hg or a reduction of diastolic blood pressure of at least 10 mm Hg within 3 minutes of erect standing.3 A transient drop that occurs with abrupt standing and resolves rapidly suggests a benign condition, such as dehydration, rather than autonomic failure.

In the laboratory, patients are placed on a tilt table in the head-up position at an angle of at least 60 degrees to detect orthostatic changes in blood pressure. In the office, 1 minute of standing probably detects nearly all cases of orthostatic hypotension; however, standing beyond 2 minutes helps establish the severity (a further drop in blood pressure).4 Orthostatic hypotension developing after 3 minutes of standing is uncommon and may represent a reflex presyncope (eg, vasovagal) or a mild or early form of sympathetic adrenergic dysfunction.4, 5
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NEUROGENIC AND NONNEUROGENIC CAUSES

Orthostatic hypotension may result from neurogenic and nonneurogenic causes.

Neurogenic orthostatic hypotension can be due to neuropathy (eg, diabetic or autoimmune neuropathies) or to central lesions (eg, Parkinson disease or multiple system atrophy). Its presence, severity, and temporal course can be important clues in diagnosing Parkinson disease and differentiating it from other parkinsonian syndromes with a more ominous prognosis, such as multiple system atrophy and Lewy body dementia.

Nonneurogenic causes include cardiac impairment (eg, from myocardial infarction or aortic stenosis), reduced intravascular volume (eg, from dehydration, adrenal insufficiency), and vasodilation (eg, from fever, systemic mastocytosis).

Common drugs that cause orthostatic hypo tension are diuretics, alpha-adrenoceptor blockers for prostatic hypertrophy, antihypertensive drugs, and calcium channel blockers. Insulin, levodopa, and tricyclic antidepressants can also cause vasodilation and orthostatic hypotension in predisposed patients. Poon and Braun,6 in a retrospective study in elderly veterans, identified hydrochlorothiazide, lisinopril (Prinivil, Zestril), trazodone (Desyrel), furosemide (Lasix), and terazosin (Hytrin) as the most common culprits.
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CLINICAL MANIFESTATIONS ARE DUE TO HYPOPERFUSION, OVERCOMPENSATION

Symptoms are related to cerebral hypoperfusion, with resulting lack of cerebral oxygenation (causing lightheadedness, dizziness, weakness, difficulty thinking, headache, syn cope, or feeling faint) and a compensatory autonomic overreaction (causing palpitations, tremulousness, nausea, coldness of extremities, chest pain, and syncope).

Lightheadedness is a common symptom, but subtler issues such as difficulty thinking, weakness, and neck discomfort are also common in the elderly. Recurrent or unexplained falls in older adults may be a manifestation of syncope due to orthostatic hypotension.
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PROGNOSIS DEPENDS ON CAUSE

Orthostatic hypotension is a syndrome, and its prognosis depends on its specific cause, its severity, and the distribution of its autonomic and nonautonomic involvement. In patients who have extrapyramidal and cerebellar disorders (eg, Parkinson disease, multiple system atrophy), the earlier and the more severe the involvement of the autonomic nervous system, the poorer the prognosis.9,10

In hypertensive patients with diabetes mellitus, the risk of death is higher if they have orthostatic hypotension.11 Diastolic orthostatic hypotension is associated with a higher risk of vascular death in older persons.12
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MANAGEMENT: FROM A TO F

The goal of management of orthostatic hypotension is to raise the patient’s standing blood pressure without also raising his or her supine blood pressure, and specifically to reduce orthostatic symptoms, increase the time the patient can stand, and improve his or her ability to perform daily activities. No specific treatment is currently available that achieves all these goals, and drugs alone are never completely adequate.

Therapies primarily consist of a combination of vasoconstrictor drugs, volume expansion, compression garments, and postural adjustment. Education about orthostatic stressors and warning symptoms empowers the patient to adopt easy lifestyle changes to minimize and handle orthostatic stress.

B
Treatment should always start with identifying and, if possible, reducing or discontinuing drugs that may be causing or exacerbating the problem (TABLE 2). Similarly, conditions that may exacerbate it (eg, anemia13) should be identified and minimized (TABLE 3).

Nonpharmacologic interventions should then be considered. They can be tried in any order or combination based on the patient’s convenience or safety. They work by expanding blood volume (taking in extra fluid and salt), decreasing nocturia (raising the head of the bed), decreasing venous pooling (wearing an abdominal binder, performing countermaneuvers, engaging in physical activity), or inducing a pressor response (drinking a bolus of cold water).

If hypovolemia is playing a major role, and the patient cannot ingest enough salt or plasma volume fails to increase despite salt supplementation, fludrocortisone (Florinef) should be considered. Untreated hypovolemia will decrease the efficacy of vasoconstrictor drugs.

Pyridostigmine (Mestinon) has a putative vasoconstrictor effect only during standing, but because its effect is modest it should be used in mild orthostatic hypotension that does not improve with nonpharmacologic measures and in moderate cases. Its effect can be enhanced with additional low doses of midodrine (ProAmatine). Midodrine with or without fludrocortisone should be used in severe orthostatic hypotension.

We use an A-to-F mnemonic to highlight management strategies (see below and TABLE 4). The alphabetic order is not meant to represent a sequential approach to management, but rather to facilitate consideration of all the available treatments.
Table 4
Table 4
Recommendations for patients with orthostatic hypotension: From A to F
A: Abdominal compression

In conditions in which there is adrenergic denervation of vascular beds, there is an increase in vascular capacitance and peripheral venous pooling. Compression of capacitance beds (ie, the legs and abdomen) improves orthostatic symptoms.14 The improvement is due to a reduction of venous capacitance and an increase in total peripheral resistance.14

On standing, healthy adults experience an orthostatic shift of approximately 500 mL of blood to the lower extremities15 that, when added to an increased vascular capacitance in those with orthostatic hypotension, results in a relative state of hypovolemia.

Compression of the legs alone is not as beneficial as compression of the abdomen because the venous capacitance of the calves and thighs is relatively small compared with that of the splanchnic mesenteric bed, which accounts for 20% to 30% of total blood volume. 16 Moreover, compression garments and stockings that are strong enough to produce a measurable effect on orthostatic hypotension are cumbersome to put on and uncomfortable to wear. Because some patients gain significant benefit from abdominal compression alone, this should be considered the first step in reducing venous capacitance.

In a laboratory experiment, Smit et al17 found that an elastic abdominal binder that exerted 15 to 20 mm Hg of pressure on the abdomen raised the standing blood pressure by about 11/6 mmHg, which was comparable to the effect of a gravity suit (such as those worn by fighter pilots to prevent syncope during violent aircraft maneuvers) inflated to 20 mm Hg—an increase of about 17/8 mm Hg. Higher gravity-suit pressures had a greater effect.

In practical terms, the binder should be tight enough to exert gentle pressure. It should be put on before rising from bed in the morning and taken off when lying supine, to avoid supine hypertension. Advantages are that a binder’s effects are immediate, its benefits can be easily assessed, and it can be used on an as-needed basis by patients who need it only during periods of prolonged orthostatic stress. Binders are also easy to fit and are available in most sporting good stores and on the Web (try searching for “abdominal binder”).

When abdominal compression alone is not enough, the addition of compression of the lower extremities can result in further benefits. This can be achieved by using compression garments that ideally extend to the waist or, at the least, to the proximal thigh.
B: Boluses of water

Rapidly drinking two 8-oz (500-mL) glasses of cold water helps expand plasma volume. It also, within a few minutes, elicits a significant pressor effect that is in part norepinephrine-mediated, 18,19 increasing the standing systolic blood pressure by more than 20 mm Hg for about 2 hours and improving symptoms and orthostatic endurance.18,20 This easy technique can be used when prolonged standing is expected (eg, shopping).
B (continued): Bed up

The head of the bed of a patient with orthostatic hypotension should be elevated by 10 to 20 degrees or 4 inches (10 cm) to decrease nocturnal hypertension and nocturnal diuresis.21 During the day, adequate orthostatic stress, ie, upright activity, should be maintained. If patients are repeatedly tilted up, their orthostatic hypotension is gradually attenuated, presumably by increasing venomotor tone.22
C: Countermaneuvers

Physical countermaneuvers involve isometrically contracting the muscles below the waist for about 30 seconds at a time, which reduces venous capacitance, increases total peripheral resistance, and augments venous return to the heart.23,24 These countermeasures can help maintain blood pressure during daily activities and should be considered at the first symptoms of orthostatic intolerance and in situations of orthostatic stress (eg, standing for prolonged periods).

Specific techniques include23:

Toe-raising
Leg-crossing and contraction
Thigh muscle co-contraction
Bending at the waist
Slow marching in place
Leg elevation.

D: Drugs

Midodrine, a vasopressor, is effective and safe when used for treating neurogenic orthostatic hypotension.25 It has been shown to increase standing systolic blood pressure, reduce orthostatic lightheadedness, and increase standing and walking time.

A common starting dose is 5 mg three times a day; most patients respond best to 10 mg three times a day. As its duration of action is short (2 to 4 hours),25–27 it should be taken before arising in the morning, before lunch, and in the midafternoon. To avoid nocturnal supine hypertension, doses should not be taken after the midafternoon, and a dose should be omitted if the supine or sitting blood pressure is greater than 180/100 mm Hg.

Midodrine’s main side effects are supine hypertension, scalp paresthesias, and pilomotor reactions (goosebumps). Vasoconstrictors such as midodrine are ineffective when plasma volume is reduced.

Fludrocortisone is a synthetic mineralocorticoid that has a pressor effect as a result of its ability to expand plasma volume and increase vascular alpha-adrenoceptor sensitivity. 28–30 This medication is helpful when plasma volume fails to adequately increase with salt supplementation31 and for patients who cannot ingest enough salt or do not respond adequately to midodrine.

The usual dose is 0.1 to 0.2 mg/day, but it may be increased to 0.4 to 0.6 mg/day in patients with refractory orthostatic hypotension.

If the patient gains 3 to 5 pounds (1.2–2.3 kg) and develops mild dependent edema, you can infer that the plasma volume has expanded adequately. However, in view of these effects, fludrocortisone is contraindicated in congestive heart failure and chronic renal failure. The potential risks are severe hypokalemia and excessive supine hypertension. Frequent monitoring of serum potassium, a diet high in potassium, and regular checks of supine blood pressure are advised, especially at higher doses, when added to midodrine, or in elderly patients who tend to poorly tolerate the medication.28,29,32

Pyridostigmine is a cholinesterase inhibitor that improves ganglionic neurotransmission in the sympathetic baroreflex pathway. Because this pathway is activated primarily during standing, this drug improves orthostatic hypotension and total peripheral resistance without aggravating supine hypertension. Because the pressor effect is modest, it is most adequate for patients with mild to moderate orthostatic hypotension.33,34

Dosing is started at 30 mg two to three times a day and is gradually increased to 60 mg three times a day. The drug’s effectiveness can be enhanced by combining each dose of pyridostigmine with 5 mg of midodrine without occurrence of supine hypertension.34 Mestinon Timespan, a 180-mg slow-release pyridostigmine tablet, can be taken once a day and may be a convenient alternative.

The main side effects are cholinergic (abdominal colic, diarrhea).

Review the patient’s medications

If he or she is taking any drug that may cause orthostatic hypotension, consider discontinuing it, substituting another drug, or changing the dosage (TABLE 2). In the elderly, antiparkinsonian, nitrate, antidepressant, diuretic, prostate, and antihypertensive medications35 may be particularly suspect.
E: Education

Education is probably the single most important factor in the proper control of orthostatic hypotension. A number of issues should be considered.

Patients should be taught, in simple terms, the mechanisms that maintain postural normotension and how to recognize the onset of orthostatic symptoms.
They must realize that there is no specific treatment of the underlying cause and that drug treatment alone is not adequate.
They should be taught nonpharmacologic approaches and be aware that other drugs they start may worsen symptoms.

It is also important that the patient learn the conditions (and their mechanisms) that can lower blood pressure (TABLE 3). Such conditions include prolonged or motionless standing, alcohol ingestion (causing vasodilation), carbohydrate-heavy meals (causing postprandial orthostatic hypotension related to an increase in the splanchnic-mesenteric venous capacitance), early morning orthostatic hypotension related to nocturnal diuresis and arising from bed, physical activity sufficient to cause muscle vasodilation, heat exposure (eg, hot weather or a hot bath or shower) producing skin vessel vasodilation, sudden postural changes, and prolonged recumbency. Once these stressors are explained, patients have no difficulty recognizing them.

The patient should also be instructed in how to manage situations of increased orthostatic stress and periods of orthostatic decompensation, to minimize nocturnal hypertension, and to modify their activities of daily living. Keeping a log of supine and upright blood pressures (taken with an automated sphygmomanometer) during situations of orthostatic stress can help establish whether worsening symptoms are related to orthostatic hypotension or to another mechanism. Once patients discover that they can actively deal with these situations, they develop a great sense of empowerment.
E (continued): Exercise

Mild physical exercise improves orthostatic tolerance by reducing venous pooling and increasing plasma volume.36 Deconditioning from lack of exercise exacerbates orthostatic hypotension.37 Because upright exercise may increase the orthostatic drop in blood pressure, training in a supine or sitting position (eg, swimming, recumbent biking) is advisable. Isotonic exercise (eg, light weight-lifting) is recommended because the incorrect straining and breath-holding during isometric exercise (eg, holding weights in the same position) may decrease venous return.
F: Fluid and salt (volume expansion)

Maintaining an adequate plasma volume is crucial. Patients should drink five to eight 8-ounce glasses (1.25 to 2.5 L) of water or other fluid per day. Many elderly people do not take in this much. The patient should have at least 1 glass or cup of fluid with meals and at least twice at other times of each day to obtain 1 L/day.

Salt intake should be between 150 and 250 mmol of sodium (10 to 20 g of salt) per day. Sodium helps with retention of ingested fluids and should be maximized if tolerated. However, caution should be exercised in patients who have severe refractory supine hypertension, uncontrolled hypertension, or comorbidities characterized by insterstitial edema (eg, heart failure, liver failure). Some patients are very sensitive to sodium supplementation and can fine-tune their orthostatic control with salt alone. If salting food is not desired, prepared soups, pretzels, potato chips, and 0.5- or 1.0-g salt tablets can be an option.

Patients need to maintain a high-potassium diet, as the high sodium intake combined with fludrocortisone promotes potassium loss. Fruits (especially bananas) and vegetables have high potassium content.

The combination of fludrocortisone and a high-salt diet can also cause sustained supine hypertension, which can be minimized by the interventions noted in TABLE 2.

Appropriate salt supplementation and fluid intake leading to an adequate volume expansion can be verified by checking the 24-hour urinary sodium content: patients who excrete less than 170 mmol can be treated with 1 to 2 g of supplemental sodium three times a day.38
Key Points

Treatment is directed at increasing blood volume, decreasing venous pooling, and increasing vasoconstriction while minimizing supine hypertension.

Patient education and nondrug strategies alone can be effective in mild cases. Examples: consuming extra fluids and salt, wearing an abdominal binder, drinking boluses of water, raising the head of the bed, and performing countermaneuvers and physical activity.

Moderate and severe cases require additional drug treatment. Pyridostigmine (Mestinon) is helpful in moderate cases. Fludrocortisone (Florinef) and midodrine (ProAmatine) are indicated in more severe cases.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2888469/

recipes for healing

hi guys, these are some recipes i have used a lot during this time… obviously, these are just very, very rough suggestions becuase all of us are so sensitive to so many different things.. so i just tried to include the basic goal of each recipe, anti inflammatory, vitamins, protien, fiber etc.. but all the ingredients are to individual taste and what is working for your body right now. i know thats so totally different for all of us. if you have recipes that have worked for you in your individual case you could please share them here for other people to use.

one of the first things that happened to me was totally losing my appetite. i couldnt handle any thought of solid food so this is why i turned to the liquid diet/smoothies. i had a smoothie almost every single day of my year long cleanse. this was a great place to add supplements or additives. i also tried very hard to get enough protein…. this came mainly from nuts, seeds, beans fish and occasional meat. again this is something that will vary with everyone. some people cant handle fruit or some kinds of fruit. i was fine with fruit i just had a problem with refined sugar. so the smoothies were a life saver for me.

my diet was fairly simple already but i -really- simplified it these past years. the cornerstones of what i survived on was fruit, nuts & seeds, yogurt, avocados, good quality corn chips (no doritos!! but corn chips without weird additives or too much oil are actually perfectly good for you) fish, beans, and vegetables like yellow peppers, broccoli, kale etc. i was ok with brown rice and potatoes in smaller amounts, and i was ok with whole wheat bread in smaller amounts. i was focusing on anti inflammatory, beneficial oils, fiber and protein, and cutting out even more any refined sugar, salt, (really had to watch the salt) and anything with additives at all, even though i rarely eat additives to begin with. i also had no problem with honey or agave syrup but i know some people do so i realize these wont apply to everyone. i also try to include seaweed in much of my cooking this is a good natural source of minerals, and use good quality sea salt rather than normal salt.

here were some recipes i used to make eating this way something tolerable:

apple #1: slice apple into wedges. (skin on) sprinkle with cinnamon to taste (no sugar!) spread with almond butter.

apple #2: chop apple into half inch chunks. (skin on) mix in bowl with cinnamon, chopped walnuts and honey.

apples are good for a lot of things, they contain pectin which can help with detoxing. apples are kind of boring by themselves so it helps to dress them up; nuts contain beneficial fats and minerals, and the cinnamon is anti inflammatory.

fruit smoothie: 1 or 2 cups fresh fruit to taste: banana, pineapple, papaya, strawberry, mango, kiwi, etc. add good quality yogurt, orange juice to taste and blend. can add nuts, seeds, tofu, for more protien. can add 1 tbsp beet powder, maca, green powder or chlorella to taste. can add greens like spinach or kale if preferred.

the goal of the fruit smoothie is:
potassium which helps balance sodium in the body (banana)
anti inflammatory and digestive enzymes (pineapple and papaya)
vitamin C (citrus, kiwi, strawberry, papaya)
good quality yogurt replenishes beneficial bacteria; it also adds protein. doesnt have to be dairy yogurt.
nuts and seeds have protein, good fats, fiber and minerals
beet powder increases oxygen availability to tissues and brain
maca is balancing to the whole neurotransmitter system

protien shake: 1 banana, yogurt, soy/almond/hemp/coconut milk as preferred; add dried fruit to taste – dried apricots, raisins, prunes, papaya, coconut (no sugar! ) add nuts and seeds to taste. (pumpkin seeds, sunflower seeds, chia seeds, hemp seeds) protien shake has more protein and dried fruit which is good for staying ‘regular’ (urr)

master cleanse: combine fresh lemon or lime juice, and (roughly equal parts) pure maple or agave syrup in a glass. add water to make a lemonade to your taste. stir in as much cayenne powder as you prefer.

the master cleanse is a very old recipe. the goal of the master cleanse is:
vitamin C (lemon or lime juice and cayenne)
increase circulation and help clear tissues (cayenne)
give the digestive system a break & cleanse the liver

edit: the real master cleanse recipe calls for pure maple syrup! i forgot this becuase i often use agave instead as it is cheaper. you can also use honey but that is considered the least beneficial.. however the real master cleanse recipe uses pure maple syrup. durr !!

fresh cilantro salsa: chop very finely (food processor if possible) several yellow or orange bell peppers; one carrot; one stalk celery; green onions; fresh garlic to taste; bunch cilantro; fresh hot chiles to taste (serrano, jalapano, habanero) add (to taste) equal parts vinegar and water to cover, juice of several limes or lemons, plus crushed coriander, black pepper, sea salt, spoon of raw sugar or agave nectar to taste. stir well and place in jars. keeps in refrigerator for a week. great with chips, on beans etc etc.

this salsa has a lot of vitamin c, garlic and onions good for the immune system, and its a good way to get a reasonable dose of fresh cilantro. i like mine really hot.. but not everyone can handle that so you can make it as not-hot as you prefer.

avocado: slice avocado in half, squeeze on lemon juice, a little sea salt and black pepper. can top with tuna or salmon salad or leave plain. also good with hummus and corn chips. (and above salsa) avocados have a lot of beneficial oils. lemon juice has vitamin c and if you can tolerate fish, tuna and salmon also contain beneficial oils.

fresh ginger spice tea: fill pot with water and add fresh ginger root sliced. simmer at a low boil for at least 1/2 hour, up to several hours is fine. the longer it simmers the more gingery it will be. add honey or agave syrup, milk or cream if desired. can also add: other whole spices (-not- powders) whole cloves, whole cardamom, pieces of nutmeg, cinnamon stick, sassafrass, licorice root, etc. can also add fresh lemon grass, fresh turmeric root, or kaffir lime leaves. all those can be gotten at good health food stores, hispanic grocery, or oriental grocery.

the goal of the ginger spice tea is basically anti inflammatory. its also a good place to add medicinal herbs like licorice or any herbs you like. dont go overboard on the whole spices! just a small amount. ginger also increases circulation and is warming if you feel cold and many spices are anti inflammatory. lemon grass is specific for the nervous system and is really delicious. also fresh turmeric root is very good if you can find it, it looks just like ginger but smaller and more orange.

these are some of the recipes ive relied on to survive and also during my year long cleanse which i am about to start year 2 of soon. i think i will just have a general cleanse every year starting on my birthday in march. spring is a good time to start cleanses. just wanted to share a few of these recipes it would be interesting to hear how you guys do it and what recipes other people have used and liked. im sure i could think of others but these were probably the most important ones for me. i know all of us are different and especially foods are different for everyone just saying what i have liked and used. take care everybody!!

Scrap of paper floating around the net.

1
8. RATIONAL DESIGN OF BOTULINUS NEUROTOXIN THERAPIES
Graham Moore*, J. Hayden*, J. Pires* and Murray G. Hamilton†
†Therapy Group, Defense Research Establishment Suffield Medicine Hat, Alberta, CANADA T1A 8K6 and
*Department of Pharmacology, University of Calgary, Calgary, Alberta, CANADA
†Author for correspondence

BOTULINUS NEUROTOXIN INHIBITORS
Short Term Goal: Discover novel inhibitors of Botulinus neurotoxin A
Long Term Goal: Design oral inhibitors of Botulinus neurotoxin A

BACKGROUND
Botulinus neurotoxin A (Botox A) is one of several protein toxins from clostridium botulinum, which cause paralytic
syndromes resulting from the blockage of neurotransmitter release. These toxins are all zinc endopeptidases acting
in the neuronal cytosol: Botox B, D, F and G as well as tetanus toxin attack specifically VAMP (also called
synaptobrevin) – a protein of synaptic vesicles; Botox A and E cleave SNAP-25 and Botox C acts on syntaxin –
both proteins of the presynaptic membrane
Botox A is a 150K protein made up of a 50K light chain (amino acids 1-448) and a 100K heavy chain (amino
acids 449-1296) which are held together by a disulfide bond (C430-C454). The heavy chain contains a
transmembrane domain (residues 659-681) which inserts into the neuronal cell membrane and enables the light
chain to access the interior of the neuron. Intracellular reduction of the interchain disulfide activates the proteolytic
activity of the light chain (zinc-binding domain HELIH, residues 223-227) which then cleaves the protein SNAP-25
and disables the docking mechanism required for exocytosis of the neurotransmitter.
SNAP-25 is one component of the so-called SNARE complex, which is responsible for docking synaptic
vesicles and fusion to the cell membrane as the immediate precursor event to transmitter release. The SNARE
complex is a four-helix bundle made up of two small proteins, namely VAMP and syntaxin, and a larger protein
SNAP-25 which doubles back on itself and provides two of the four threads of the helix bundle. SNAP-25 also
contains a lipid anchor region (amino acids 85-92) between the two helical threads, which lines up with
transmembrane domains at the C-termini of both VAMP and syntaxin (Poirier et al, Nature Structural Biology
5:765,1998). Whereas Botox A selectively cleaves the Gln187-Arg203 bond near the C-terminus of SNAP-25, the
other botulinus neurotoxins each selectively cleave a different peptide bond within one of the three target proteins
which comprise the SNARE complex.
A repeating motif exists within the sequences of all three of the proteins of the SNARE complex which, when
introduced in the form of synthetic peptides of about ten residues in length, inhibits the actions of botulinus toxins
on the SNARE complex (Rossetto et al, Nature 372: 416, 1994). For example one version of the repeating motif
present in VAMP, designated V2 and having the sequence 62ELDDRADALQ71, blocks the neurotoxic actions of
Botox A and B when the peptide is injected into cultured Aplysia neurons. The significance of the repeating SNARE
motif, which appears twice on each of the four threads of the SNARE helix bundle upstream of the cleavage sites, is
not well understood. Presumably it acts as a recognition site for binding of some other biomolecule(s), and may also
be used by Botox as a binding-recognition element. In agreement with this, cross recognition of the target proteins
by the various toxins occurs: Botox A inhibits VAMP proteolysis by Botox B, and Botox B and tetanus inhibit the
cleavage of SNAP-25 by Botox A. Moreover serum albumin, which contains SNARE motifs within helical regions
of its secondary structure (183DELRD187 and 255DDRAD259), inhibits the cleavage of synthetic substrate by
Botox A.
Thus although the mechanism of action of V2 (and other variants of the SNARE motif) is not proven, it could
involve binding to a complementary recognition site on Botox resulting in inhibition of productive binding of Botox
to the SNARE complex. Interestingly, possible complementary SNARE motifs (592KKVNK596 and
701KRNEK705 in the heavy chain, and 335KLKFDK340 and 359KVLNRK364 in the light chain) exist in Botox A.
STRATEGY
Several possible strategies for inhibiting Botox exist:
• Block the transmembrane domain of Botox and prevent cell entry, e.g. complementary peptides, antibodies;
• Block the proteolytic site of Botox, e.g. substrate inhibitors, selective chelation of zinc;
• Block the interaction of Botox with binding-recognition motif, e.g. mimics of SNARE motif, which bind to
complementary site(s) on Botox.
2
The first strategy has potential application prior to exposure to toxin (e.g., immunization), as well as for the
deactivation of toxin in body fluids after exposure. The latter strategies, on the other hand, have the potential for
providing total body treatment after toxin exposure – assuming that the therapeutic agent is able to cross cell
membranes and inactivate toxin which has already entered the cell. Whereas agents based on active site inhibitors
will have to be tailored to individual variants of Botox, therapies based on the SNARE motif have the potential to
treat poisoning by all forms of Botox as well as tetanus.

Active sites of zinc proteases
The most widely studied of all the zinc proteases is carboxypeptidase A (CPA), and information on the
mechanism of action of this enzyme serves a useful basis for understanding other related zinc enzymes such as
Botox.

Detailed X-ray studies on CPA have illustrated that the tetradentate zinc atom sequesters a single water
molecule and is tethered by coordination to the imidazole groups of His-69 and His-196 as well as to the carboxylate
of Glu-270. CPA undergoes a conformational change on binding of the substrate Gly-Tyr, in which the guanidinium
group of Arg-145 moves 2A to form a salt bridge with the C-terminus of the substrate, the carboxyl group of Glu-
270 moves 2A away from the zinc atom and forms a salt bridge with the amino group of the substrate (can only
occur with dipeptide), and the phenolic group of Tyr-248 moves 12A to within 3A of the scissile bond (Reeke et al,
PNAS 57, 2220, 1967). Chemical modification studies have suggested that Tyr-248 is essential for the peptidase
activity, but not the esterase activity, of CPA. A catalytic mechanism has been proposed in which the susceptible
carbonyl oxygen of the substrate coordinates to zinc and the Glu-270 carboxylate attacks the carbonyl carbon to
form a mixed anhydride intermediate, which is subsequently hydrolyzed by a base catalyzed mechanism involving
the nascent water molecule (Zn-OH).
The best inhibitors of CPA are generally compounds that coordinate with the active site zinc and either
sequester the zinc atom away from the enzyme or prevent access of the substrate to the catalytic center. These
include thiols, amines/imines, flavonoids, and a range of anionic groups including carboxylates, phosphonates, etc.
The most potent inhibitors will be those having an exact fit to the active site of the enzyme which is accompanied by
strong attractive forces resulting in high affinity binding not only to the zinc atom but also to the enzyme’s peptidic
groups.
Due to its preference for basic amino acid residues, the enzyme most like Botox A is carboxypeptidase B. CPB
differs from CPA in that it contains three disulfides (CPA has only one) and the coordination of zinc involves a free
Cys thiol. Exchange of zinc for cadmium in CPB results in only esterase activity (no peptidase activity).
Replacement of Asp-253 (which normally binds the basic sidechain of the substrate) with Lys gave a reversedpolarity
mutant of human CPB which hydrolyzed hippuryl-L-glutamic acid (Edge et al, Protein Eng. 11, 1229,
1998).
The best inhibitors of CPA are benzyl- or alkyl-succinic acids and, assuming some parity between CPA and
CPB, likely inhibitors of CPB would be aminoalkyl- or guanidinoalkyl-succinic acids. For reasons of specificity, it
follows that those considerations which apply to CPB will likely also apply to Botox A.
Accordingly, peptide
libraries directed towards the active site of Botox A should be rich in basic and acidic moieties. Interestingly, acidic
residues are also a characteristic of the SNARE motif, so that peptide libraries composed of these residues should
interact with both the catalytic site and the SNARE motif site and act as dual inhibitors.
Regardless of mechanism, one approach to producing improved inhibitors of Botox is to identify the structural
elements of V2 (and other SNARE motifs) that make it an effective inhibitor. Then reconstruct these components
into a smaller and preferably nonpeptidic molecule, which would be able to traverse membranes, thereby providing
not only access to the inside of cells, but also the potential for oral activity. A streamlined and efficient approach to
this goal is to create a library of small semimimetic peptides containing the essential elements of the repeating motif
in the SNARE complex, identify the most active peptide in the mixture by iterative deconvolution of the library, and
then restructure the best semimimetic peptide identified into a fully fledged nonpeptide mimetic using computer
molecular modeling techniques.
The important structural feature of the SNARE motif is comprised of an amino acid sequence made up of
residues:
A-A-x-x-A-x-x
Where A = acidic residue and x = nonpolar or polar residue.
Due to the helical arrangement of these groups within the secondary structure of the SNARE proteins from
which they are derived, the end result is a cluster of three neighboring negative charges juxtaposed by a nonpolar
moiety. In other words, the required motif can be envisaged as three negatively charged groups, mounted in close
proximity on a hydrophobic template. The simplest representation of this that comes to mind, is a benzene ring with
three carboxymethyl groups attached at the 1, 3 and 5 positions. Other variations on this theme can also be easily
3
envisaged, and a number of commercially available compounds that fit this general scheme are being investigated
for inhibitory activity in our laboratory, together with several peptide libraries which have been designed and
synthesized with these considerations in mind.
DESIGN AND SYNTHESIS OF PEPTIDE LIBRARIES
One library has been synthesized which is designed to mimic the SNARE motif:
SN-1 = Ac -X1-X2-LINKER-X3-X4-NH2,
Where X1, X2, X3 & X4 are mixtures of Asp, Glu, Gln and Arg, and the LINKER group is 4-aminobutyric acid
The rationale for the design of the peptide minilibrary SN-1 is based on the preponderance of acidic residues in
SNARE motifs, together with the occurrence of other residues in SNARE motifs which can provide solubility
properties to the libraries – hence the inclusion of Arg and Gln in SN-1. The multiple negative charges represented
by the Asp and Glu residues, when mounted around the flexible linker group 4-aminobutyric acid (Abu), should
engender structural and conformational variations on the SNARE motif which will inhibit Botox.
Coincidentally, the Gln and Arg residues in SN-1 are also representative of the scissile bond (Q-R) in the Botox
A substrate, so that the peptide minilibrary SN-1 doubles in a rudimentary way as a substrate mimic. This parallels
the classic approach to inhibitor discovery, which is usually done by minor modification of substrate structure
leading to compounds that bind to the active site but are not cleaved by the enzyme. Accordingly, incorporating
residues of the scissile bond (Gln-Arg) into peptide libraries such as SN-1 should provide structural and
conformational characteristics that might result in substrate-based inhibitors. Clearly SN-1 contains the potential for
containing inhibitors based not only on the SNARE motif but also on substrate structure.
Another consideration for the design of peptide libraries with inhibitory potency is the inclusion of residues
which would be expected to coordinate to the active site zinc atom of Botox, namely Cys and His as well as acidic
residues. The approach here is to create a peptide library containing an array of zinc binding elements in a variety of
different formats, wherein there should be individual peptides which recognize not only the zinc atom but also
unique aspects of the active site of Botox A in which the zinc atom resides. The intent is to produce zinc targeted
inhibitors that show selectivity for Botox over other zinc metalloenzymes.
An example of a library targeted to produce active site zinc inhibitors is:-
SN-3: Ac-X1-X2-Abu-X3-X4-NH2
where Ac = acetyl, Abu = 4-aminobutyric acid, and X1, X2,. X3, and X4 are mixtures of the amino acids Asp,
Glu, His and Cys.
This library contains both active site zinc directed probes and variations on the SNARE motif within the
same basis set (library) of peptides.
CONFORMATIONAL PROPERTIES OF TARGET BINDING SITES
Before intricate design work on inhibitors of Botox can be undertaken, details of the three-dimensional
conformation of the target sequence must be elucidated. In the case of Botox A, the substrate SNAP-25 is believed
to form part of a four-helix protein bundle that is anchored into the presynaptic membrane. Actually the cleavage
site targeted by Botox A is near the C-terminus of SNAP-25, which is far removed from the central lipid anchor
region of SNAP-25. Similarly the amino acid sequences representing the four SNARE motifs of SNAP-25 (S1, S2,
S3 and S4) reside in the central part of the SNARE complex away from the lipid anchor domain found at one end of
the tubular bundle. Accordingly, it is not clear if the environment surrounding the sequences representing not only
the cleavage site but also the SNARE motifs is primarily polar (aqueous) or nonpolar (membrane lipid) in character.
As a precautionary measure we have conducted conformational calculations on these target sequences firstly in
a nonpolar environment and subsequently in a polar environment. Semiempirical energy minimization calculations
carried out on the substrate SNAP-25 (187-203) revealed that this peptide takes up a helical conformation in a
nonpolar environment (in vacuo), but that the introduction of water molecules into the environment results in the
helix unraveling. Accordingly, the conformation of this peptide is highly dependent on environment, although the
spatial arrangement of amino acid sidechains in the critical region of the QR scissile peptide bond remains the same.
It is not clear if Botox A is likely to preferentially recognize either the helical conformation or the disrupted
conformation, and at present there is no way of knowing which of the two conformations would be appropriate for
design work on potential active site inhibitors.
Likewise, semiempirical calculations on the SNARE motifs V2 and S3 have revealed helical structures in a
nonpolar environment which become disrupted in the presence of water. Again, since there is no way of knowing
which is the biologically relevant conformation, it is not possible to conduct design work based on the information
presently available. Consequently we have elected to proceed with peptide library investigations in the hope that the
identification of a bioactive semimimetic peptide will provide the answer to this question. Thus, eventually it should
4
be possible to identify from library screening, a potent inhibitory ligand containing the essential structural elements
of the SNARE motif, which is sufficiently conformationallly restricted that it can only take up one of the two
SNARE motif conformations – either that found in membranes or that found in water. At that stage it will then be
possible to use the appropriate conformational model for further detailed design work.
OPTIMIZATION OF ASSAY CONDITIONS
Several laboratories have reported assays of Botox A activity against various substrates. Rossetto et al (1994)
investigated the action of Botox A against intact SNAP-25 and found that only the reduced form of the enzyme
(pretreated with 10mM DTT for 30 min at 37C) could cleave the substrate. For the subsequent incubation with
substrate, they used 5mM HEPES buffer pH 7.4 for 60 min at 37C, without the addition of supplementary zinc.
These authors also reported that the enzyme was blocked by 10mM EDTA (zinc chelator) or by 1.4mM captopril
(zinc endopeptidase inhibitor).
Schmidt & Bostian (1997) studied the activity of Botox A towards a number of short synthetic substrates
derived from the cleavage site of SNAP-25 and found that peptides of at least 15 residues were required for cleavage
by the reduced form of the enzyme. Incubations were carried out in 30mM HEPES buffer pH 7.3 in the presence of
5mM DTT, 250uM zinc and 1mg/ml BSA for 10 min. at 37C. Activity against these substrates required the presence
of BSA and increased with increasing concentrations of BSA to a maximum effect at 1mg/ml BSA. The activity was
blocked by 5mM EDTA or 100mM Tris (zinc chelators).
In this laboratory we have investigated the effects of pH and varying concentrations of zinc and DTT on the
activity of the reduced enzyme. Reduction was accomplished by incubating the enzyme with 20mM fresh DTT at
37o for 30 min. in 50mM HEPES pH 8. After reduction optimal activity against a 17mer peptide from SNAP-25
(residues 187-203) was observed by preincubating the enzyme (5nM) in 15mM HEPES buffer pH 6.9 in the
presence of 1uM zinc and 0.5mM DTT for 5 hr before adding the substrate (50uM). After 30 min. the reaction was
terminated with an equal volume of 1% TFA and the products determined by HPLC. The activity was inhibited in
the complete absence of zinc or in the presence of zinc concentrations above 10uM, and was also inhibited by
10mM DTT. These findings illustrate that Botox A, like many other zinc endopeptidases, requires the presence of
zinc but is inhibited by excess zinc. In addition, since high concentrations of DTT can inhibit, possibly due to the
zinc chelating properties of DTT, a careful balance between zinc and DTT concentrations must be engendered.
INHIBITORS OF BOTOX A
In our studies a number of compounds were found to inhibit the Botox A mediated cleavage of the 17-amino acid
synthetic peptide substrate Ac-SNKTRIDQANQRATKML-NH2, which derives from the C-terminal part (residues
187-203) of SNAP-25 and contains the scissile QR peptide bond targeted by Botox A (Table 1). Several thiolcontaining
compounds, namely dithiothreitol (widely used to reduce disulphide bonds in proteins), DMPS (a potent
chelator of heavy metals) and Captopril (a clinical inhibitor of the zinc dipeptidase Angiotensin Converting Enzyme)
block the cleavage of this substrate. However the non-thiol ACE inhibitor Lisinopril was ineffective, whereas the
non-thiol prodrug ACE inhibitor enalapril was an effective inhibitor.
The thiol compounds DMPS, captopril and
DTT presumably act by a mechanism involving sequestration of the zinc atom at the active site of Botox A. The
inhibitory activity of enalapril was unexpected but could suggest an affinity for the active site of Botox A which is
not shared by lisinopril.
The synthetic peptide V2 was also able to inhibit Botox A, but at a higher concentration (5mM) than has been
observed previously in vivo when SNAP-25 was the substrate (100uM, see Rossetto et al 1994). This may reflect the
absence of a SNARE motif in the short synthetic substrate used for the present studies.
Interestingly serum albumin (10uM), which contains the SNARE motif in duplicate, inhibited cleavage of
SNAP-25 (187-203) under our assay conditions which had been optimized for zinc (1uM) and DTT (0.5mM)
concentrations. In contrast Schmidt & Bostian (J. Protein Chem. 16: 19, 1997) observed that in the presence of
250uM zinc and 5mM DTT cleavage of this substrate by Botox A only occurred in the presence of BSA. Our
findings would suggest that this was probably due to sequestration of inhibitory levels of zinc/DTT by BSA.
Investigations of SN-1 on the Botox A cleavage of substrate has shown that this peptide minilibrary does inhibit
the enzyme activity (Table 1). This suggests that variations on the SNARE motif present in the library mixture may
interfere with binding of Botox to the substrate. However a series of peptides which were selected for their potential
to represent simple variations on the SNARE motif, i.e. Glu-Glu, Glu-Glu-Leu, Glu-Glu-Glu and Glu-Pro-Glu-Thr,
were generally inactive (data not shown), with the notable exception of Glu-Glu-Glu. A number of peptides with
potential complementary sequences of the SNARE motif, i.e. Lys-Arg, Lys-Lys, Orn-Orn, Lys-Lys-Lys, Orn-Orn-
Orn and Lys-Phe-Gly-Lys, were inactive as expected because the synthetic 17mer peptide substrate used for the
assay lacks the repeating SNARE motifs that are present in the longer SNAP-25 natural substrate. However these
5
basic peptides would be expected to inhibit the cleavage of SNARE proteins by Botox enzymes in general, if they
bind well to the SNARE recognition motifs in these proteins.
Interestingly, glycyrrhizic acid, which is a steroid glycoside containing 3 carboxylate groups, was able to inhibit
Botox (Table 1). This finding, as with Glu-Glu-Glu, suggests that certain configurations of negative charges are able
to approximate the SNARE motif and inhibit Botox A.
However in the final analysis it would appear that the most
potent inhibitor of Botox containing a SNARE motif variation is likely to be found in the peptide library SN-1
because each peptide in the mixture was present at 20uM concentration (Table 1).
Finally, the present experimental findings also indicate that the best inhibitors of Botox A are likely to be
derived from thiol containing compounds (the inhibitory activity of DMPS is particularly remarkable). In agreement
with this, the peptide minilibrary SN-3 turned out to be an exceptionally potent inhibitor of Botox A (Table 1).
When the overall thiol concentrations of the inhibitors are compared (Table 1) it is apparent that the peptide library
SN-3 is the most potent thiol inhibitor of Botox. Furthermore it is very likely that SN-3 contains a peptide(s) which
is a more potent inhibitor than DMPS.
We are currently evaluating SN-3 inhibition of other related zinc protease neurotoxins namely Botulus B and
tetanus neurotoxin. The intent is to deconvolute the library and determine which peptide in the mixture is the best
inhibitor of Botox A and which peptide in the library is the best inhibitor of Botox B and tetanus. The expectation is
that there will be sufficient differences in the active sites of the toxins to yield different optimal inhibitors from the
same ‘hinge’ peptide minilibrary.
ACKNOWLEDGEMENTS
This research work was supported by an award from the Defense Research Establishment Suffield to G.M.
REFERENCES
1. Poirier et al: Nature Structural Biology 5:765 (1998).
2. Rossetto et al: Nature 372: 416 (1994).
3. Schmidt & Bostian: J Protein Chem 16: 19 (1997).
4. Moore GJ: Drug Devel Res 42: 157-163 (1997).
5. Moore GJ: Trends Pharmacol Sci 15: 124-129 (1994).
6. Reeke et al Proc. Nat. Acad. Sci. USA57: 2220 (1967).
7. Edge et al Protein Eng. 11: 1229 (1998).
FIGURES AND TABLES
SNARE Complex: Lateral
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SNARE Complex: Top View
Details of SNARE complex, showing the substrate, V2 and S3
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Table 1. Compounds found to inhibit the Botox A mediated cleavage.
Inhibitor Concentration (mM) % Inhibition* -SH Concentration (mM)
Dithiothreitol 1 26 2
DMPS‡ 0.1 72 0.2
Captopril 1 76 1
Lysinopril 5 7 0
Enalapril 5 40 0
V2 (ELDDRADALQ) 5 40 0
Glu-Glu-Glu 5 22 0
Glycyrrizic acid 5 55 0
Library SN-1 5 (20 μM)† 16 0
Library SN-3 0.5 (2 μM)† 51 0.125

Between today and yesterday.

It’s so weird.

Yesterday I was nonfunctional, absolutely. I had muscle pain, the shakes, total stupidity, inability to focus in the EXTREME. I ended up just laying down and just staring for hours, then around 2 AM suddenly felt a little better. This morning I woke up and have had three hours of being “normal.” When I am normal, I start letting a stray thought or two fly around about plans for the future and I try to get “thinking work” done.

But what was the difference? I can’t figure it out. Now I am starting to get botoxy again but I would rather have three hours of normal than a whole day of total incapacity.

I wish I could figure it out.