To Awake a Sleeping Beauty

Every year over 1,000 hospital admissions for accidental poisoning, attempted suicide, or drug overdosage occur in this city and there is no doubt that the size of this problem is increasing. A large variety of drugs are taken and the specific treatment of the more common poisonings is adequately dealt with in an excellent monograph of Mathew and Lawson. It will help however to consider some general principles of how to tackle the acute problem clinically. You can: (1) Prevent the drug entering the body’s circulation (stomach washouts) (2) Prevent the drug reaching its site of action once in the body (3) Prevent the drug from exerting its effect at this site by (a) direct competitive inhibition (b) providing alternative metabolic pathways to overcome drug action (4) Enhance the rate of drug metabolism (5) Remove the drug from body— (diuresis, dialysis). Of these methods, the first, and the last to a lesser extent, is widely used in practice in this country. Again I refer you to Mathew’s book for the details. I am interested in the third of these, that is to prevent the drug from exerting its effect at its site of action by providing alternative metabolic pathways, with particular reference to barbiturate poisoning, which constitutes the largest single group of agents encountered in clinical practice. Copyright Royal Medical Society. All rights reserved. The copyright is retained by the author and the Royal Medical Society, except where explicitly otherwise stated. Scans have been produced by the Digital Imaging Unit at Edinburgh University Library. Res Medica is supported by the University of Edinburgh’s Journal Hosting Service: http://journals.ed.ac.uk ISSN: 2051-7580 (Online) ISSN: 0482-3206 (Print) Res Medica is published by the Royal Medical Society, 5/5 Bristo Square, Edinburgh, EH8 9AL Res Medica, Spring 1971, 6(5): 24-26 doi:10.2218/resmedica.v6i5.874 T O A W A K E A SLEEPING BEAUTY John Wallwork, B.Sc. Every year over 1,000 hospital admissions for accidental poisoning, attem pted suicide, or drug overdosage occur in this city and there is no doubt that the size of this problem is increasing. A large variety of drugs are taken and the specific treatment of the more common poisonings is adequately dealt with in an ex­ cellent monograph of M athew and Lawson. It will help however to consider some general principles of how to tackle the acute problem clinically. Y o u can : (1) Prevent the drug entering the body’s circulation (stomach washouts) (2) Prevent the drug reaching its site of action once in the body (3) Prevent the drug from exerting its effect at this site by (a) direct com petitive inhibition (b) providing alternative metabolic pathways to overcome drug action (4) Enhance the rate of drug metabolism (5) Rem ove the drug from body— (diuresis, dialysis). O f these methods, the first, and the last to a lesser extent, is widely used in practice in this country. Again I refer you to M athew ’s book for the details. I am interested in the third of these, that is to prevent the drug from exerting its effect at its site of action by providing alternative m etabolic pathways, with particular reference to barbiturate poisoning, which constitutes the largest single group of agents encountered in clinical practice. Before we can antagonise a drug’s action in this way we must know where that site of action is, and how the drug works. L et us look at a little simple biochemistry. T h e main biochemical pathway for the pro­ duction of energy is the metabolism of glucose. Represented in a simplified form. Glycolysis -> Krebs cycle E.T. Chain —> Energy. Theories concerning the m ode of action of barbiturates on this system have varied. Early work by Brody and Bain suggested that the action of barbiturates was mediated by an un­ coupling of oxidative phosphorylation. How­ ever Aldridge and Parker (1960) showed that inhibition of respiration occurred w ithout un­ coupling, in liver mitochondria; more import­ ant from our point of view, they also showed that the inhibition produced by barbiturates on the respiration of mitochondria, did not occur when sucoinate was used as substrate instead of glucose. It was concluded that the block must occur somewhere before the entry of succinate into the metabolic process. Elaborate techniques used by Chance and Hollinger in 1965 corroborated this work. Imagine the process as a production line with substrate in a reduced form at the left and products in oxidised forms to the right. If we stop this continuous process somewhere in the middle, there will be a build up of re­ duced substances to the left and a depletion of oxidised substances to the right of the block. T h e substances involved can be measured in both their oxidized and reduced state in the presence o f a barbiturate in the metabolic pathway. Therefore the site of block in the chain can be determined by the point at which the transition between elev­ ation of the reduced proportion and depletion of the oxidized proportion of such substances occurs. This is then the site o f action of the barbiturate.


T O A W A K E A SLEEPING B EAUTY
John Wallwork, B.Sc.
Every year over 1,000 hospital admissions for accidental poisoning, attem pted suicide, or drug overdosage occur in this city and there is no doubt that the size of this problem is increasing.
A large variety of drugs are taken and the specific treatment of the more com m on poisonings is adequately dealt w ith in an ex cellent monograph of M athew and Lawson.It w ill help however to consider some general principles of how to tackle the acute problem clinically.Y o u can : (1) Prevent the drug entering the body's circulation (stomach washouts) (2) Prevent the drug reaching its site of action once in the body (3) Prevent the drug from exerting its effect at this site by (a) direct com petitive inhibition (b) providing alternative m etabolic pathways to overcome drug action (4) Enhance the rate of drug metabolism (5) R em ove the drug from body-(diuresis, dialysis).O f these m ethods, the first, and the last to a lesser extent, is w idely used in practice in this country.Again I refer you to M athew 's book for the details.
I am interested in the third of these, that is to prevent the drug from exerting its effect at its site of action by providing alternative m etabolic pathways, w ith particular reference to barbiturate poisoning, w hich constitutes the largest single group of agents encountered in clinical practice.
Before we can antagonise a drug's action in this way w e must know where that site of action is, and how the drug works.
L e t us look at a little sim ple biochemistry.
T h e main biochem ical pathway for the pro duction of energy is the m etabolism of glucose.Represented in a simplified form.
Theories concerning the m ode of action of barbiturates on this system have varied.Early work by Brody and Bain suggested that the action of barbiturates was m ediated by an un coupling of oxidative phosphorylation.H ow ever Aldridge and Parker (1960) showed that inhibition of respiration occurred w ith ou t un coupling, in liver m itochondria; more im port ant from our point of view, they also showed that the inhibition produced by barbiturates on the respiration of m itochondria, did not occur when sucoinate was used as substrate instead of glucose.It was concluded that the block m ust occur somewhere before the entry of succinate into the m etabolic process.Elaborate techniques used by C h an ce and Hollinger in 1965 corroborated this work.
Imagine the process as a production line w ith substrate in a reduced form at the left and products in oxidised forms to the right.If we stop this continuous process somewhere in the m iddle, there w ill be a build up of re duced substances to the left and a depletion of oxidised substances to the right of the block.
T h e substances involved can be measured in both their oxidized and reduced state in the presence o f a barbiturate in the m etabolic pathway.
T herefore the site of block in the chain can be determ ined by the point at w hich the transition between elev ation of the reduced proportion and depletion of the oxidized proportion of such substances occurs.T h is is then the site o f action of the barbiturate.T h is is very satisfying biochem ically, bu t all this work was done in vitro, a long w ay from the com plicated structure and m echanism of w hole brain.D oes barbiturate have the same action in w hole brain?W e designed an experim ent on th e hypo thesis that barbiturates have the same site of action in vivo and in vitro.
I t involved anaesthetising rats with a barbiturate and giving them a substrate, m etabolised in brain, w hich enters the pathw ay before the proposed site of action (we used pyruvate), and one that enters after the proposed site o f action (we used succinate).W e observed.
T h is sounds easy.H ow ever, the difficulties in adm inistering substances to th e brain in known concentration are great.
T h e sub stances we used, pyruvate, succinate, are utiliz ed b y other tissues in the body; they are also highly ionized and 'therefore w ill not easily cross the so-called blood-brain barrier.
R ecen tly new techniques have been develop ed to overcom e such difficulties, som e o f great sophistication; we used a rather crude but effective technique.
O nce the rat is anaesthetized by an intraperitoneal injection of barbiturate, a sm all hole is bored at a particular p o in t on the skull.Substances are then injected in sm all volum es into one of the lateral ventricle o f the brain, through a special needle so designed th at the depth from guard to tip is the depth required to reach the lateral ventricle in rats of a certain weight.
U sing this technique the experim ent was elaborated using three groups o f rate o f the same age, sex and weight.O ne group received standard dose of barbiturate alone, one group barbiturate and intraventricular succinate and

II oxidised
the other group barbiturate and intra ventricular pyruvate.T h e sleeping times of all rats, that is the tim e from onset of anaes thesia to tim e of arousal, were determ ined.
O ther variables were elim inated by using (1) R ats of the same strain and w eight and receiving a standard dose o f barbiturate I.P. (2) Starved rats, so that absorption from peri toneum would be m ore predictable.
(3) M ale rats since B rody has shown that bar biturates have different lengths o f action in different sexes.T h e results were striking.T h e re was no significant difference in the control group and the group given pyruvate; both groups having sleeping times o f around 10 0 m inutes.H ow ever, the group receiving succinate had a mean sleeping tim e of around 30 m inutes.T h is is a h ighly significant difference.
It can be argued that succinate in som e way decreased the concentration o f available bar biturate either by increasing its m etabolism or by inactivating it.In order to elim inate this possibility, samples o f blood were collected from all rats by decapitation at the tim e of arousal.
T h e blood barbiturate levels were estim ated.T h e levels in the blood o f the succinate group killed at around 30 m inutes were found to be significantly h igher than the levels in the control and pyruvate group killed around 100 m inutes.T h is was to be expected if the succinate did not affect the barbiturate blood level and it was concluded th at the action o f succinate m ust be central in the brain.
(Intra cellular oxidation at reversal level).
From these results we can postulate that our original hypothesis was correct since, like the m itochondria discussed earlier, the rats could utilize succinate for energy production, to overcom e the barbiturate anaesthesia, but were not able to utilize pyruvate.
W e therefore concluded that the barbiturates act in the sim ilar w ay in whole brain as they do in isolated m itochondria.
T h e question now arises as to w hether the action o f barbiturates cannot be reversed in hum ans, if succinate can reverse barbiturate anaesthesia in rats.T h is seems to b e a good idea, but I have already m entioned difficulties due to blood-brain barrier effects, w hich would exclude the possibility of using succinate intravenously.In fact two workers, Soskin and Taubenhous, tried this, with no effect, in 1943, long before Kregs invented his cycle.I t is difficult to persuade anybody that bor ing holes in hum an heads to inject directly into brain tissue is of justifiable therapeutic value.A nd the present m ortality rate after hospital admission is sm all, due to the exten sive supportive therapy em ployed in overdose treatment.
It has been suggested that we perform cysternal punctures in particularly ill patients bu t this is a difficult and not w idely practiced technique and would be available only to the few.
W e m ust use an intravenous route of ad m inistration if this m ethod o f treatm ent is to be useful and we m ust therefore m odify the succinate m olecule in som e way.It occurred to us that the answer m ight be to make a fat-soluble succinate m olecule with the active site free.It could enter the brain and still act as a substrate for m etabolism .A substance that appeared to have the required physical properties was m onoethylsuccinate, highly soluble in water and organic solvents.Finding a recipe to make this substance proved to be more difficult.H owever, we now have a whole 50 grams and hope to begin work soon.T h e theory is that this substance w ill enter brain in sufficient concentrations and will be split by esterases to give free succinate and, incidentally, ethyl alcohol to bring you round drunk.
As I have already indicated, barbiturate poisoning is a m ajor problem .M u ch tim e and m oney and m any lives could be saved if every doctor in the land could wake these sleeping beauties with a sim ple injection.
T h ere is a long way to go.T h is is only the beginning.
Chance & H ollanger.J. Bio. Chem . 278, 418 (1963) C h an ce and H ollinger showed a build up of reduced N A D P H 2 and a depletion o f oxid ized cytochrom e b in the presence of bar biturate and concluded that its site o f action m ust be between N A D P H 2 and F A D cyt.b (Site I in diagram ).I f the site of action had been site I I then they w ould have observed an accum ulation of reduced cyt.b and depletion of oxidized cyt.c. concluded that barbiturates pre vent respiration of liver m itochondria by in hibition of the m etabolism at the site shown (N A D P H2/cyt.b).