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Periodicity of Influenza

THE LANCETLONDON: SATURDAY, APRIL 13, 1946

EPIDEMIOLOGISTS have spilt much ink in the effortto show that influenza epidemics recur in, some sortof regular sequence. Most famous are BROWNLEE’S

attempts to demonstrate an underlying 33-week cycle.Earlier workers, however, were handicapped in thatthey did not know of the existence of two differentagents, influenza viruses A and B, antigenicallyunrelated but each capable of producing widespreadoutbreaks. The United States commission on acuterespiratory diseases has lately been analysing thesequence of outbreaks in-America since 1920 in the

light of present knowledge. They take the excessannual death-rates from influenza and pneumoniain 90 cities and plot them week by week over 25 years.The peaks occur with bewildering irregularity. Thecurve is then analysed on the assumption that theoutbreaks are all caused by either virus A or B,influenza A having a cycle of periodicity of 2-3 yearsand influenza B a longer one of 4-6 years. There now

appears something approaching regularity, as thetable shows.

The left-hand part of the table is wholly conjecturalso far as it implies that one or other virus was a causeof trouble, for neither virus was recognised before1933. The guesses are, however, reasonable ones,considered as extrapolations from data obtained inmore recent years. The right-hand part of the table ismore trustworthy, for the viruses concerned were (apartfrom the American " A " record of 1933) actuallyidentified. It will be noted that the A outbreakswere in step on the two sides of the Atlantic. Britishworkers have emphasised that some epidemics (thosein parentheses were minor ones, and the figures fromAmerica show that there also the 1933, 1937, and1943 outbreaks were larger than the intervening ones.This winter we have been sharing with our Americanfriends the experience of a B outbreak, but hithertothere has been less evidence of correspondence betweenthe two countries in epidemics of influenza B than ofA. So far as we know, B has never been very trouble-some in this country before this winter. Are we now any nearer forecasting the future 1

Not, we fear, very accurately. The U.S. commissionthought that the chances favoured an A outbreak in

1. Amer. J. Hyg. 1946, 43, 29.

the winter of 1945-46 and a widespread outbreak ofB "before the summer of 1946." According to

present information, their forecast was wrong as

regards A but right as to B. If their interpretation oftheir curves is correct, both viruses were behavingfairly regularly between 1928 and 1941, B turningup every fourth year and A every other year in thatperiod, the epidemics never coinciding. Since 1941there has been less stability, with fortunately a

tendency to less frequent manifestations of activityby either virus. It would be justifiable to lay oddsagainst a recurrence of B next winter, but the outlookas regards A is obscure. We may, however, have apointer before the autumn. In 1943 scattered smalloutbreaks of A during the late spring and summerled up to the autumn A epidemic. Similarly B inAmerica gave evidence of its activity in the summerof 1945 before becoming widely prevalent in the

following winter. Localised occurrences of flu in thesummer months are clearly worth watching.

Chemistry of the PenicillinsINTENSIVE research on the chemistry of the peni-

cillins has been carried out during the war on bothsides of the Atlantic, but in 1943 a publication banon all this work was imposed by the British andAmerican governments. The ban is now being lifted.To coordinate the chemical investigations on peni-cillin in Britain, the Medical Research Council set upa committee, representing the various academic andindustrial groups involved, and the correspondingwork of chemists in the U.S.A. was coordinated bythe Office of Scientific Research and Development.Since March, 1944, a complete exchange of informa-tion between the British and American groups hasbeen established. Recently a preliminary statementof the main results of this collaborative Anglo-American effort was published simultaneously inS’cience 1 and Nature,2 and it is hoped that a fullaccount will be published in the form of a comprehen-sive Anglo-American monograph later this year.The most important result of the Anglo-American

work is that the chemical structure of the penicillinshas been completely elucidated. They are dipeptidesof a special type, possessing a common nucleus butdifferent side-chains. Their general structural formulais as follows :

(CH3)2 . C--CH. COOH where R varies in the

’ a 2

I I different penicillins, So

ks N far four different peni-B / B cillins have been obtainedCH CO in the pure crystallineB / state. In England theyCH were designated as peni-

_ NH CO R cillins I, II, III, and m,NH. CO. Pt, in accordance with the

historical order of their discovery ; in the U.S.A.they were termed F, G, x, and K. In penicillin

’ I (F) B -is 2-pentenyl CH,CH2 - CH=CH . CH2-11 (G) benzyl C6H5CH,-

111 (x) "" p-hydroxybenzyl OH . C6 H4 . CH2- -iv (K) ’ n-heptyl CHaCH2CH2CH2CH2CH2CH2-

The probable structure of the penicillin molecule wasderived by chemical degradation and synthesis of

1. Science, 1945, 102, 627.2. Nature, Lond. 1945, 156, 766.

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model compounds. Finally it was proved conclusivelyby crystallographic X-ray studies on single crystalsof the potassium and rubidium salt of penicillin n,which led to accurate measurements of the bonddistances between all the atoms of the penicillinmolecule.The unique feature in the penicillin formula is the

four-membered " (3-lactam " ring, a structure so far

not encountered in any other biological material.This four-membered ring is opened easily by alkali,primary alcohols and amines, certain thiol compounds(e.g., cysteine), and the enzyme penicillinase which ispresent in some naturally resistant bacteria. This

ring fission is the cause of the high instability of thepenicillins towards these reagents. The open chaincompounds, formed by fission of the four-membered(3-lactam ring, are thiazolidines which have beentermed penicilloic acids. By the action of alkali andpenicillinase on sodium penicillin the salts of the

penicilloic acids are formed ; methanol inactivationof penicillin produces mono-methyl esters, inactivationby benzylamine mono-benzylamides of the penicilloicacids.

With mercuric chloride the penicilloic acids (or theirderivatives) are decomposed into a thiol amino-acid"

penicillamine "-(CH3 )2 . C CH. COOH

SH NH2

(&bgr;-dimethyl-cysteine) and aldehydo-acids, " penaldicacids " (or their derivatives). The free penaldic acids,being &bgr;-carbonyl acids, are unstable and decomposespontaneously into carbon dioxide and aldehydes,termed " penilloaldehydes

"

Penicillamine, the constituent of the penicillinmolecule common to all penicillins, belongs to theseries of amino-acids with the unnatural d configura-tion. The structure of penicillamine, the penicilloicacids and many of their derivatives, and derivativesof the penaldic acids and the penilloaldehydes haveall been proved by unequivocal synthesis as well as bydegradation.One of the most characteristic chemical properties

of the penicillin molecule is the ease with which

it undergoes intramolecular rearrangement. ’Thisfaculty of rearrangement is illustrated in the two

following examples :(1) It is well known that the penicillins rapidly lose

their antibacterial power when kept for a short timeand at room temperature in a weakly acid medium. Thisbiological inactivation of the penicillins by acid is dueto the formation of isomeric compounds termed " penil-lic acids." The penillic acids are converted by alkaliinto " isopenillic acids," and by mercuric chloride into. substances containing a free SH group and termed" penillamines." The penillic acids, isopenillic acids, andpenillamines are imidazol derivatives all of which havebeen synthesised. (See formula A on opposite page.)

(2) The methylester of penicillin 11 is transformed intothe isomeric methylester of " penicillenic acid " by theaction of mercuric chloride at room temperature innon-polar solvents. The penicillenates contain the five-membered azlactone ring, and on alkaline hydrolysisafford the sodium salt of 2-benzyl-4-hydroxymethyleneoxazolone. (See formula B on opposite page.)

These two rearrangements are very unusual andcould hardly have been predicted from the knownreactions of organic chemistry. The amide groupssuch as are present in the side-chains of the peni-cillins are known to be relatively non-reactive ; yetin the penicillin molecule they can react underextremely mild conditions to form an imidazol or anoxazolone ring. These reactions are of considerable

general biochemical interest.As might have been expected from their biological

properties the penicillins have proved to bean entirely novel type of molecule, not

resembling any known synthetic or naturalantibacterial product. Their structure is

relatively simple : they are peptides made upof two simple amino-acids, dimethylcysteine

and an acylated serine, the alcoholic group of whichhas been oxidised to the corresponding aldehyde.Through the incorporation in a novel manner of apeptide linkage into a four-membered lactam ring,the molecule has acquired its unique biological andchemical properties. This is, indeed, an astonishingfact which no chemist could ever have predicted. Thepeptide nature of the penicillin molecule makesunderstandable its complete non-toxicity to animaltissues. One is naturally tempted to ask whether theoccurrence of a 3-lactam ring in nature is limited

exclusively to penicillin or whether this configurationmay not possess a broader biochemical significanceoccurring also in other biologically active peptidesand proteins. _

<

Despite the apparent simplicity of the penicillinmolecule it has not so far been possibleto achieve a workable method of syn-thesis, although many efforts to thisend have been made by competentchemists. However, the production ofpenicillin by the natural fermentationprocess has now become so cheap that

tne syn1!neSlS 01 penicillin is a promem oi more

academic than practical importance. It may in factbe difficult, if not impossible, ever to devise a syn-thetic process which -can compete successfully withbiological production.Whenever a new type of chemotherapeutic agent

is discovered one of the main aims of the chemistsis to improve its properties by chemical modificationof the molecule. In the absence of a synthesis for thecomplete penicillin molecule the only possibility for

541

getting modified penicillin thathas to be considered at present is,chemical alteration of the naturalpenicillins. From their chemicalstructures it would appear thatpenicillins I, n, and IV do notlend themselves to any chemicalmodification of their molecules.Penicillin ill, however, possessinga p-hydroxybenzyl side-chain,would seem to offer considerablescope for chemical modification by introduction ofnew groups into the side-chain. It would be desirableto improve the properties of the penicillins in threedirections : (1) to increase the range of susceptiblebacteria ; (2) to increase their stability ; and (3) toreduce their rate of exeretion. , Can such improve-ments be achieved by modification of the side-chains inthe penicillin molecule ? The evidence available fromthe studies of the four natural penicillins with differentside-chains suggests that with regard to (1) and (2)there is little prospect of success. All four penicillinsattack the same type of bacteria and only minor

quantitative differences in -their bactericidal powerhave been observed; they all possess roughly thesame degree of stability towards acid and alkali andthe enzyme penicillinase. With regard to (3), however,it would seem possible that systematic work on theintroduction of suitable groups into penicillin ill maylead to modified semi-synthetic penicillins, that areexcreted less rapidly than the original natural pro-ducts. To achieve success in this line of work it willfirst be necessary to develop fermentation methodswhich will make penicillin m more plentiful than itnow is. A promising advance in this direction waslately reported by K. B. RAPIER and D. F. ALEXANDER

of the U.S. Department of Agricultureto the American Association for theAdvancement of Science. By exposinga culture of P. notatum to ultravioletradiation they have isolated a newstrain which gives a high yield ofpenicillin with at least half of it inthe x or ni form. The best yieldof penicillin in from strains previouslytested was a fifth of the total. Itis not yet certain that the change in

the fermenting capacity of the new strain is the resultof the irradiation, but this seems probable. -

It is clear that an outstanding piece of work hasbeen accomplished which has brought to light manynew facts of absorbing chemical and biochemicalinterest, extending far beyond the limited field ofpenicillin chemistry.

Cardiac CatheterisationIN 1929 FoRSSMANN had a ureteric catheter passed

through the basilic vein into his own right auricle.Two years later, MONIZ, DE CARVALHO, and LIMA 2were using the method in Lisbon for the injection ofradio-opaque material into the right heart to outlinethe vessels at the lung’ root ; and in Paris in 1936AMEUTLLB and his colleagues 3 applied the techniqueto 60 patients. All this was done without mishap,but in each case the catheter was in position for onlya few minutes. In 1941 COURNAND and RANGES,4 4

basing their confidence on animal experiment, hadthe courage to pass catheters into the right auricle in1. Forssmann, W. Klin. Wschr. 1929, 8, 2085.2. Moniz, E., de Carvalho, L., Lima, A. Pr. m&eacute;d. 1931, 39, 996.3. Ameuille, P., et al. Bull. Soc. m&eacute;d. H&ocirc;p. Paris, 1936, 60, 729.4. Cournand, A., Ranges, H. A. Proc. Soc. exp. Biol., N.Y. 1941,

46, 462.

man and to leave them in situ for an hour or so whileobservations were made on the effect of variousinfluences on the heart. The method enables pressures in the right heart to be measured, and, byanalysing the gas content of blood from the rightheart, the cardiac output can be calculated by theFick method. Following CouBNAND’s leadership asteadily increasing number of schools are now employ-ing this remarkable method in cardiovascular studies.In June, 1945, under COURNAND’S chairmanship, theAmerican Physiological Society held a symposium oncardiac output 5 in which it was announced that over1200 cardiac catheterisations had been done inAmerica and England without mishap. The methodseems to be safer than either lumbar puncture -orgastroscopy, and to carry no more risk than the

5. Cournand, A. Fed. Proc. 1945, 4, 183, 207.