Wound healing

WOUND

HEALINGt

AN EXPERIMENTAL STUDY OF WATER SOLUBLE C~OROPH~L DERIVATI~S CONJUNCTION WITH VARIOUS ANTIBACTERIAL AGENTS

IN

LAWRENCE W. SMITH, M.D. AND ALFRED E.LIVINGSTON, PH.D. PHILADELPHIA, PENNSYLVANIA

I

N

a previous report, certain preliminary experimental studies concerning the effect of the water-soIubIe derivatives of chIorophyIl* upon wound heahng were presented. It was noted that an overall acceleration of heaIing of about 25 per cent in time was obtained in somewhat over two-thirds of the experimentaIIy induced Iesions with the chIorophyI1 preparations as compared with any of the other twelve test agents and the bases empIoyed in the study. The experimental Iesions which were studied in rats, guinea pigs, rabbits and dogs were of three types: first, clean, surgicaIIy induced wounds; second, experimentaIIy infected surgicaIIy induced wounds; and third, standardized dry heat, third degree burns. Breaking down the statistical data, it was found that with the chIorophyII preparations the highest acceIeration percentage figures were obtained in the case of clean wounds and burns as we11 might have been anticipated. In specific experiments this acceleration figure ran as high as 83 per cent and in no instance feI1 beIow 55 per cent. By contrast in a comparabIe group of cIean cases using the other test agents and controls with their bases, the percentage figures of acceIerated healing range from o to 50 per cent with an overa average of about 20 per cent, but with a time factor of less than IO per cent. Indeed, delay in heaIing, especiaIIy with the suffa drugs, was ~ommonIy encountered

in these cases, the percentage varying from 5 to 67 per cent. On the other hand, in the chIorophyI1 treated group of cIean, surgicahy induced wounds, no delay .in heaIing was noted except in five or six cases in which secondary wound contamination developed as the resuIt of the animal getting his dressing off. AnaIysis of the figures from the experimentaIIy infected wounds in these initial studies emphasizes the vafue of suppIementing the wound heaiing effect of chIorophyI1 by some antibacteria agent with a wider effective range than chIorophyI1 alone possesses. It has been shown “in vitr0”2 that chIorophyI1 has defmite bacteriostatic properties, and under suitable environmenta circumstances apparentIy, even some bactericida1 capacity; but the action of chIorophyI1 seems to be more in the nature of enhancing tissue resistance by reIative acceleration of the norma repair mechanism, and perhaps by creating an unfavorabIe milieu for bacteria1 survivai, than by any direct action on the bacteria themseIves. This was evidenced by the fact that even in the presence of wound infection acceIeration of heahng was noted in 63 per cent of the induced Iesions treated by chIorophyI1 aIone. With the suIfa preparations alone the picture shifted from delayed healing in over 50 per cent of the clean wounds to acceleration of the repair process in nearIy 30 per cent of the infected Iesions. The same shift was noted in the series treated by the organic iodine compound, tetrodine, from a delay of about 20 per cent in the clean

* The chIorophyI1 preparations used in these studies have been generousIy supplied us through the courtesy of the Rystan Company, New York City, sole appointee of the Lakeland Foundation of Chicago.

j From the Departments of Pathology and Pharmacology, Temple University SchooI of Medicine, Phiiadefphia, Pa. Aided by a grant from the Committee on Therapeutic Research of the CounciI on Pharmacy and Chemistry of the American Medical Association, Chicago III.

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NEW SERIES VOL. LXVII.

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Livingston-Chlorophyll

wounds to actuaI acceIeration in 45 per cent of the infected Iesions. The encouraging resuIts obtained in our

American

Journal

ofsurgery

31

(2) the saving of extremities, (3) the hastening of sIoughing of necrotic tissue, (4) the acceIeration of heaIing and (5) the

FIG. I.

preIiminary studies with these watersoIubIe chIorophyI1 preparations on the healing of burns and infected wounds led us to beIieve that further investigation in this fieId, using chIorophyII* in conjunction with some of the newer antibacteria1 agents might prove even more fruitfu1. That the satisfactory treatment of burns and infected wounds stiI1 remains one of the major problems not onIy of the war but also of civihan Iife can scarceIy be denied. Thus, any contribution, no matter how small, which tends to better the resuIts obtained by our present admittedIy inadequate methods of treatment, merits serious consideration, The uItimate aims of any treatment used in such types of injury might be Iisted as: (I) the reduction of mortaIity, *Whenever

the term “chIorophyI1” is used in this the water-soluble derivatives are meant, the term “chIorophyI1” being used solely for the sake of brevity.

paper,

reduction in disfiguring cicatrix production by the promotion of normaI, soft scar formation. In addition, the saving of time with its economic implications, Iikewise shouId be stressed. It was beIieved that our earIier studies had dehniteIy estabIished the vaIue of “chIorophyI1” as a therapeutic agent in its own right, through its stimuIating effect upon normal fibroblastic proliferation,3 in the healing of burns and infected wounds when treated topicaIIy. We had empIoyed the chIorophyI1 derivatives both in saIine soIutions as wet dressings and in various ointment and jeIIy bases, without significant differences in respect to the acceIeration of the heaIing time of the various experimental lesions. In clinica studies the wet dressings were found particuIarIy usefu1 during the first twentyfour to ninety-six hours in cleaning up grossIy contaminated wounds. The physica characteristics of the hydrophiIic jelly type

32

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Livingston-ChIorophyIl

of base were not entireIy satisfactory as it tended to dry out, causing the dressing to become adherent and painfu1 unIess changed frequently. The oIeophiIic type base had the famiIiar objection of mereIy forming an emoIIient, insuIating Iayer which did not permit adequate contact of the drug to the actua1 wound surface. Thus, we uItimateIy obtained through the co-operation of various agencies* an ‘yidea1 ” ointment base which combined the features of being sufficientIy emoIIient to resist drying out for periods of a week to ten days, whiIe at the same time being sufEcientIy hydrophiIic to reIease the medicaments incorporated in its substance at a fairIy steady, slow rate. In addition severa “dusting-powder” types of preparations were Iikewise used for comparison in the studies. This search for an “ideaI” base invoIved the testing of severa intermediate preparations on guinea pigs before the actuaI investigation couId get under way. In aI1, somewhat over 200 experimental Iesions were’ observed during the period of their heaIing, haIf of them treated by I per cent chIorophyI1 in the particuIar base under consideration, the other haIf serving as controIs and treated by the base alone. The great majority of these were cIean infection ocwounds, gross secondary curring in somewhat Iess than IO per cent of the lesions. AcceIeration of healing of the chIorophyI1 treated Iesions, as compared with the contro1 Iesions, by approximateIy 25 per cent in time was found in this series in 71 per cent of cases as compared with 68 per cent in the earIier reported cases. Lest the question be raised as to whether the effective agent in these preparations is actuaIIy the chIorophyI1 derivatives and *We wish to acknowledge our indebtedness to Dr. A. 0. WhiDDle and Dr. E. L. Howes of CoIumbia Universitv,‘f;lew York City, for suggestions as to the general c&position of a universallysatisfactory base, and to Isidor ChameIin. Goldwater MemoriaI Laboratories, Welfare Island, %&York City, Research Fellow of the Lakeland Foundation, Chicago, for the preparation of the ointment bases used in these studies.

JaNU*RY, 1949

not some component of the particular vehicIe, some thirty additiona controIs were run, using simpIe boric acid or petroIatum on the Iesions on one side and the severa ointment bases, incIuding the base finaIIy adopted, on the other. No difference in the rate of heaIing was noted under these comparative conditions other than minor variations of a day or so in individua1 guinea pigs. With these preIiminary background studies serving as controIs for the ointment bases, simiIar experiments were conducted using the I per cent “chIorophyI1” ointment in combination with various antibacteria1 agents. Owing to Iimitations of time our efforts were IargeIy concentrated on certain of the suIfa drugs, peniciIIin and tetrodine, as representing three of the most generaIIy accepted types of such therapeutic drugs. Two per cent of the foIIowing suIfa compounds were incorporated in the chIorophyI1 ointment: (I) suIfaniIamide, (2) suIfathiazoIe, (3) sulfadiazine, (4) suIfamerazine, (5) sulfasuxidine and (6) a new experimental product of LederIe’s-carboxy-suIfathiazoIe. In addition, peniciIIin was empIoyed in the same manner in a concentration of 250 Oxford units per Gm. FinaIIy, tetrodine powder of ointment. was added to the “chIorophyI1” ointment to give the recommended strength of 6 per cent free and combined iodine to the preparation with the object of comparing the effect of a true chemica1 antiseptic with the antibiotic action of the other substances. The entire study was carried out on guinea pigs, with the exception of a few experiments using dogs for comparative purposes. Twenty-four animaIs were used in the experiments with each drug. TWO symmetrica Iesions approximateIy I cm. in diameter were made on opposite sides of the abdomen without any attempt at maintaining surgica1 asepsis. The wounds were inoculated with a IoopfuI of a twentyfour-hour broth cuIture of a stock hemoIytic staphyIococcus (c-zag), covered with a

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I

Smith,

Livingston-ChIorophyII

simple wet broth dressing to encourage bacteria1 growth and prevent the animaI from Iicking the Iesions cIean. The foIIowing day treatment was begun. The Ieft hand Iesion in each instance was treated with the chlorophyII-antibacteria ointment. The right hand control Jesions were divided into three groups of eight each. One group was dressed with the I per cent chlorophyll ointment alone, a second group with the ointment base aIone, and the third group with the antibacterial agent in the ointment base but without chlorophyll. By this means it was believed that a11 possible sources of confusion in respect to the several factors involved could be eliminated. An initial tracing of the Jesion was made and simiIar measurements recorded each time the wounds were dressed at two- or three-day intervaIs until healing was compIete. No effort was made to determine anything but this one end point ---the healing time required in each instance-in much the same practicaJ way in which the great majority of cIinica1 cases are treated and their progress noted. Blood Ievels of the drugs were not noted except in occasional isoIated instances. The resuIts of these wound healing studies are summarized in Table I. At first gIance, it might appear that the differences expressed by these “ average ” figures are not sufficiently marked to be of much cIinica1 significance. However, on more careful examination, it becomes apparent that certain differences do exist, which tend to follow a fairly well defined pattern. In the first place, it has been shown that I per cent of the aqueous soIubIe chIorophyJ1 derivatives added to the ointment base acceIerates the healing process in the care of wounds to an appreciabIe degree. It shouId be emphasized that this acceIeration is even ‘more marked in the larger Iesions as observed in the dog experiments and in cJinica1 cases. In other words, the “chIorophyI1” itseIf gives evidence of having a moderate antibacteria action in the presence of infection, as we11

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d Surgery

as of a stimuIating effect upon the wound healing mechanism as shown in the earIier studies comparing the heaIing rate in lesions treated by base aIone and by the base with added chIorophyIJ. TABLE I .A\‘EKAC;E VI MHFKOF DAYSREQL i11’4LIYG

OF

: IUFECTED

EXPERIXIEiSTALLY WITH

STAI’UYLOCOCC~ 4s

TREATED

STRAIN

IRED

I\

TOPICALLY

BY

CIIEMOTHERAPEUTIC

GUINEA VARlOl

PIGS S

AGEUTS

Control Series Test Agent in Ointment

NO. Le_

sions

f2%)

CO~II’LE’,‘,~. WOU*uDS

C-209 HEMOLYTlC

S PYOGEUES)

Chlorophyll ointment alQne PeniciIIin (250 units per Gm). SulfaniIamide ia(i;i Sulfathiazolc (2~~) SuIfadiazine (2yc,!, SuIfamerazine (25;). Sulfasuxidine (27;). CarboKy-suIfathiazoIe

FOR

INDUCED

No.

Days

.1

Test Series Test Agent in 1%

NO. Lc_

sions

No.

‘~Days

58 8 6 8

. . . . ..I

'4.3

IS.0

24

10.6

16.2

22

‘5.3 14.8 16.2 16.0

21

19 23

‘3-4 ‘3-5 13.7 I$.0 16.0

14.8

23

~ 1r.0

20

(

_

Total--AH sulfa compounds.............. Tetrodine (674, iodine).

42

lj.j

'4.9

128 22

13.5 ‘3.3

WhiIe the average acceIeration with the chIorophyI1 ointment has been shown to be about 23 per cent in time there have been wide variations in individua1 cases, in some instances as much as 40 and 50 per cent. In the second place, in no instance was the average time required for heaIing of the lesions in any of the experiments in which the suppIementary antibacteria agent was combined with the ointment base alone as satisfactory as with the chIorophyl1 ointment aIone. Indeed, actua1 deIay in heaIing was observed in the case of certain of the suIfa drugs, notabIy suIfaniIamide, suIfamerazine and suIfasuxidine. The individua1 cases show con-

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siderabIe Auctuation in this respect, possibly due to the particuIar animal’s constitutiona susceptibihty to the drug,

FIG. 2. Note chlorophyI1 treated Iesion (A) entireIy healed with controt (B) showing Iittle evidence of repair.

or possibly due in some instances to concomitant infection by suppIementary secondary suIfa-resistant organisms, but with deIay of as much as 50 per cent in time in some instances. When we tur.n to a consideration of the experiments in which the antibacteria agent is added to the chIorophyI1 ointment there are severa interesting observations to record. It wiI1 be noted that of a11 the drugs tested, peniciIIinQ in the strength of 250 Oxford units per Gm. of the chlorophyI1 ointment, gave the most briIIiant resuhs. The time required- for compIete healing was reduced from the 14.3 days needed with straight I per cent chIorophyI1 ointment, and fifteen days for the base and penicillin aIone to an average of 10.6 days with the chIorophyI1 peniciIIin * The penicilIin used in these studies was the sodium saIt prepared by the Charles Pfizer Company and reIeased to us for experimental purposes through courtesy of Dr. Chester S. Keefer, Chairman bf Committee on Chemotherapeutic Agents of the tiona1 Research CounciI.

was the the Na-

JANUARY, rg.u

combination, an additional saving of approximateIy 33% per cent in time. In this group there were six animaIs in which healing occurred within eight days. Despite the instabiIity of peniciIIin, we found that the potency of the drug was reduced onIy negIigibIy over a forty-eight-hour period, if the ointment mixture was kept in the refrigerator. (This particuIar ointment base, containing no water, is particuIarIy we11 adapted for topical use with peniciIIin.) (Fig. 2.) Analysis of the resuIts obtained with 2 per cent of th e various suIfa drugs incorporated in the chIorophyI1 ointment yielded interesting resuIts. The figures 13.4, 13.5, and 13.7 days, respectiveIy, to secure complete healing with the sulfanilamide, suIfathiazoIe and suIfadiazine preparations pIus chIorophyI1 are so nearly identical that it wouId be impossible to determine any differences in their therapeutic effxciency by this means. The healing time is reduced somewhat in a11 three instances as compared with that of the lesions treated by chIorophyI1 ointment aIone. The difference, however, is not striking as in the case of peniciIIin. With the suIfamerazine and sulfasuxidine in combination with the chIorophyI1 ointment the same deIay was noted in the heaIing of the experimenta Iesions, as was observed when these sulfa drugs were incorporated in the ointment base aIone. This wouId suggest that these drugs are perhaps more irritating when topicahy appIied, or perhaps their sIower absorption might expIain the deIay in controIIing the infection. At al1 events on the basis of these particuIar experiments they wouId not appear to be the sulfa drugs of choice for topicaI therapy in such cases. On the other hand, a new experimental suIfa compound, tentatively designated as 5-carboxy-suIfathiazoIe, * was found to be aImost as effective as peniciIIin when used topicahy in the same combination with chIorophyI1 ointment. In twenty-three *This product was placed at our disposal through the courtesy of the Lederle Laboratories, New York.

NEW SERIES Var. LXVII,

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animaIs the time required for compIete heaIing of the Iesions with this ointment was cut down from 14.3 to I 1.0 days, or approximateIy 30 per cent. This particuIar suIfa compound appears to have a very Iow toxicity, a sIow soIubiIity and has a hydrogen ion concentration approaching neutraIity. TABLE

-

I

Healing ir Days

i z 3 4

~Ointment aIone Ointment alone Ointment ! alone ~Ointment aIone

base

28

base

25

base

31

I y_ chIorophyIl ointment

HeaIing in Days

1;

21

ointment I y0 chlorophyll ointment I ‘+4echlorophyll ointment

Test Agent Left side

I % chIorophyI1 ointment I y0 chlorophyll ointment c‘%suIfaniIamide ointment 2 y. carboxy-suIfathiazole ointment 2 To carboxy-suIfathiazole ointment 2 y0 suIfaniIamide ointment I y0 chIorophyI1 2 5% ,” SuIfaniIamide ointment I % chIorophyI1 2 y. carboxy WIfathiazole

base . 27

I yo chlorophyI1

II

~ 21 23

I 21

19 23 3‘4 28

base aIone-average 27.9 days. chIorophyI1 ointment average 2 I .9 days.

New York.

35

infecting organisms, and permitting healing to proceed at the acceIerated rate noted in the other cases, under the influence of the chIorophyI1 factor. STUDIES

WITH

CHLOROPHYLL

DUSTING

POWDER

A small series of animaIs were studied in the same way using chIorophyI1 as a dusting powder. The chIorophyI1 was miIIed to 200 mesh fineness and combined in I per cent concentration with Iactose, caIcium lactate, suIfaniIamide, carboxysuIfathiazoIe, carbowax 4000 (80 mesh), and with gIycine and boric acid both aIone and with Tetrodine. One animaI was used as a contro1, using the agent alone, without the chIorophyI1. Three animaIs were treated with chIorophyI1 mixtures. It is our impression that none of these preparations is as satisfactory as the ointments. DOG

30

21

18

I ‘%

FinaIIy tetrodine, * a water-soIubIe organic iodine product which we used in our previous studies, and which we have adopted routineIy as a skin antiseptic in our experimenta operative anima1 work, was empIoyed in a simiIar manner, in conjunction with the chIorophyI1 ointment. Owing to the necessary Iow hydrogen ion concentration of the iodine preparation the resuItant mixture of the two substances is not permanently stabIe, but by combining them freshIy each time the animaIs were dressed, it was possibIe to carry out the studies satisfactoriIy. It wiI1 be noted that the iodine acted in its usua1 capacity as a chemica1 germicide, destroying the * The Tetrodine was generousIy supplied us by the TyIer Laboratories,

of Surgery

27

N. B. Ointment

American Journal

EXPERIMENTS

SuppIementing the guinea pig experiments eight dogs were simiIarIy treated by producing two symmetrica 7 cm. wounds in their backs. We were interested particuIarIy in noting the satisfactory physica characteristics of the chIorophyI1 ointment, when applied to wounds which were more nearIy comparabIe in size to those encountered cIinicaIly, with especial reference to the fact that it did not dry out and retained its emoIIient features when dressings were reappIied onIy at five- to seven-day intervaIs. In addition we were concerned with the comparative rate of heaIing in the Iarger type Iesion, treated with chIorophyI1 ointment aIone and with the suIfa compounds added. Obviously, it is impossibIe to draw any definite concIusions from such a smaI1 number of animaIs, but it seems significant perhaps that the resuIts conform in genera1 to those observed in the Iarger series using guinea pigs. TabIe II gives the essential data reIating to these dog experiments. It wiI1 be noted that the best result in time was obtained with the Iesion

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treated by the chIorophyII-carboxy-suIfathiazole combination as was true in the guinea pig lesions. This was foIlowed

JANUARY, 1945

in position to fit the individua1 animal’s neck. This represented a modification and a distinct improvement over the corrugated cardboard coIIar previousIy described by us.l The coIIar did not interfere with the animal’s eating, drinking, or lying down. Indeed, the dogs became accustomed to wearing the coIIars within a very short time, and were not bothered by them in the Ieast. The dressings were heId in pIace IocaIIy by adhesive suppIemented by a jacket made of unbleached cotton sheeting or toweIing with hoIes cut for the foreIegs and then sewed in place on the back as previousIy described. The same technic was employed in dressing the guinea pigs and proved to be most satisfactory. COMMENTS

FIG. 3. IlIustrates the method of protecting dressings on dog’s back by speciaI pIywood colIar.

very cIoseIy on an eIapsed time basis by the Iesions treated by chIorophyI1 ointand the combination of ment aIone, chIorophyI1 and suIfaniIamide. The Iesion treated by the carboxy-suIfathiazoIe ointment aIone and the controIs on which the ointment base aIone was used paraIIeIed each other. There was definite deIay in the healing of the two Iesions treated by sulfanilamide. ointment aIone. The ointment base itseIf proved to be entireIy satisfactory physicaIIy, retaining its emoIIient features for as Iong as a week. We believe it is particuIarIy we11 adapted for cIinica1 use. Very much the same method was empIoyed in these dog experiments as in the guinea pig studies. The wounds were intentionaIIy contaminated to simuIate actual cIinica1 conditions. The chief probIem was to keep the animaIs from getting their dressings off. This was accompIished by means of a wide colIar (Fig. 3) made of Iight pIy-wood, with an adjustabIe wedge which couId be wired

The experimenta evidence presented in these studies appears to speak for itself and requires very IittIe discussion. It suppIements our earIier studies in which the acceIerating effect of chIorophyI1 upon wound heaIing was compared most favorabIy with that obtained by a number of other therapeutic agents rather commonIy empIoyed for that purpose. However, because of the reIativeIy narrow range of the effectiveness of chIorophyI1 as an antibacteria agent, but because of its outstanding tissue growth-stimulating effect in wound healing, it was believed it might prove worth whiIe to expIore the possible advantages of combining some of the newer, more potent antibiotics with chIorophyI1 in the topical treatment of wounds. These hopes appear to have been fuIIy justified by the resuIts obtained in these experimenta studies. CriticaI anaIysis of the data serves to bring out and confirm severa more or Iess seIf-apparent facts. It adds to the rapidIy accumuIating evidence of the tremendous vaIue of peniciIIin in the treatment of infected wounds by topica appIication of the drug, as reported by CIark et aI.,” FIorey and Cairns5 and many others.‘jv7v8 It simiIarIy emphasizes the perhaps’ Iess

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spectacuIar but equaIIy important pIace of the sulfonamides in the same lieId, suppIementing the ora or parentera administration of the drugs. The apparent reIationship of the reIative soIubiIity of the suIfonamides to their effectiveness in their topica appIication was brought out with great cIarity. The particuIarIy good resuIts obtained with the new experimenta1 carboxy-suIfathiazoIe member of the sulfa family Ieads us to beIieve that it shouId be given serious consideration in the cIinica1 heId, and made the object of thorough investigation of its pharmacoIogic action. These studies further co&m our favorable consideration of Tetrodine as a skin antiseptic. Its compIete soIubiIity in water, Ieading to deeper tissue penetration, and its remarkabIe Iow tissue irritabiIity, coupIed certainIy with an equa1 bactericida1 effect to that of tincture of iodine make it an outstanding substitute wherever tincture normaIIy is used. FinaIIy, these studies augment the data which we have accumuIated regarding the pIace which “ chIorophyI1” seems destined to fiI1 in our therapeutic armamentarium in the treatment of burns and wounds. AI1 the evidence which has accumuIated from anima1 experimentation seems to point practically incontrovertibIy to the growth stimuIating effect of chIorophyI1 upon the deveIopment of heaIthy granuIation tissue with concomitant acceIeration of heaIing of both cIean and infected wounds, and with the added advantage of the production of soft scar tissue instead of dense cicatrix formation. Furthermore, as shown both by in vitro and in vivo2’3 chIorophyI1 appears to exert a definite bacteriostatic effect in its own right. H owever, in the case of grossIy contaminated wounds, especiaIIy those caused by the pyogenic cocci, the addition of penicilIin or certain of the sulfa drugs to the chlorophyI1 preparations results in a noteworthy shortening of the time required for the eIimination of the infection, and of the compIete heaIing of the lesion.

American

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In the experiments which we have recorded in this paper, this acceIeration of the whoie repair mechanism by the combination of chIorophyI1 with such antibiotic agents gives us the impression of being what might be termed a symbiotic rather than mereIy expressing response, the simpIe cumuIative effect of the two agents independentIy. The possibiIity that the chIorophyI1 acts in some unexpIained manner as a cataIyst in the process is suggested as a reasonabIe expIanation of the phenomenon, but one which wiI1 require further expIoration experimentaIIy to estabIish. ConcomitantIy with these Iaboratory studies certain cIinica1 observations have Iikewise been made, a11 of which tend to confirm the resuIts obtained in the anima1 experiments and those aIready reported in the Iiterature. Among the latter shouId be cited the papers of Buergi,g Boehringer,1° Gruskin,ll Gahan et aI,l* WaIIace13 and GoIdberg. l4 Mention shouId aIso be made of HoImes and MueIIer’s15 successfu1 use of chIorophyI1 ointment in the treatment of burns incidental to x-ray therapy. These water soIubIe chIorophyI1 preparations have been used in a wide range of cIinica1 conditions. Their greatest vaIue seems to be in three or four genera1 lieIds of therapy, although because of the broad physioIogica1 basis of their action, many other types of disease might equaIIy we11 be benelited. Thus far, from the pubIished Iiterature aIready cited, and the ‘cIinica1 appIications of its use with which we are familiar, it seems as if they had proved to be particuIarIy usefu1 in nose and throat work, especiaIIy the treatment of chronic sinus disease; in the denta held in the treatment of Vincent’s stomatitis and periodonta1 disease (pyorrhea aIveolaris), and in the treatment of burns and wounds. In this Iatter lieId the most striking results have been obtained in Iong standing chronic cases which have been found stubbornIy resistant to a11 other forms of treatment.

38

American

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Jawmu,

Livingston-ChIorophylI

It has been the priviIege of one of us (L.W.S.) to aid (directIy, or indirectIy in an advisory capacity to a number of our friends) in the treatment of a number of such chronic resistant cases. These have incIuded severa Iongstanding cases of osteomyeIitis, some of severa year’s duration; at least three burn cases which had faired to hea after as much as eighteen to twenty months previous therapeutic efforts, and a dozen or more cases of indolent uIcers, chieff y of periphera1 vascuIar origin. In many of these the resuIts have been aImost dramatic. SeveraI extremities for which amputation had been urged have been saved, and compIete healing has taken pIace in unbeIievabIy short periods of as IittIe as tweIve to twenty days in a few instances. In the denta fieId, the resuIts appear to have been equaIIy dramatic. GoIdberg,14 in his paper emphasizes the importance of the water-soIubIe chIorophyI1 derivatives in the treatment of ora sepsis, especiaIIy Vincent’s stomatitis, in which he reports chIorophyI1 aImost a specific. James,* of Temple University DentaI SchooI, confirms GoIdberg’s findings in a series of some 200 cases of periodonta1 disease. SUMMARY I. A preIiminary study of chIorophyI1 in various ointment bases in over 200 experimentaIIy induced Iesions in guinea pigs showed acceIeration of heaIing of approximateIy 25 per cent in time in 71 per cent of the chIorophyI1 topicaIIy treated Iesions as compared with those treated by the ointment base aIone. 2. Further studies upon a series of 192 guinea pigs with biIatera1, symmetrica1, infected, surgica1 Iesions I .o cm. in diameter simiIarIy treated topicaIIy with I per cent chIorophyI1 ointment combined with various of the suIfa drugs, with peniciIIin, and with Tetrodine were carried out. 3. Twenty-four animaIs were used in the experiment with each antibacteria being divided into agent, the controIs * Persona1 communication-unpublished data.

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three equa1 groups of eight animaIs; onethird being treated with base aIone, onethird with base pIus chIorophyI1 aIone, and the fina third with base pIus antibacteria1 agent aIone. 4. The topica use of peniciIIin in strength of 250 Oxford units per Gm. of ointment combined with I per cent chlorophyI1 gave the most spectacular resuIts, with heaIing compIete ifi 10.6 days as compared to 14.3 with chIorophyI1 alone, and 15.0 days for peniciIIin aIone, an acceIeration of nearIy 35 per cent. 5. The suIfa compounds, in combination with chIorophyI1, in genera1 showed some acceIeration of heaIing, through their contro1 of infection which appeared to be roughIy proportiona to their soIubiIity. 6. Carboxy-suIfathiazoIe was by far the most effective of these suIfa compounds, nearIy equaIIing peniciIIin in that respect. 7. SuIfaniIamide, suIfamerazine and suIfasuxidine aIone in the ointment base actuaIIy caused some deIay in healing. 8. Tetrodine empIoyed in the same manner was of about the same order of effectiveness as suIfaniIamide, suIfathiazoIe and suIfadiazine. g. A smaI1 series of guinea pigs treated by chIorophyI1 combined with various agents as a dusting powder gave Iess cIearIy defined resuIts and further studies do not seem warranted. IO. Experiments carried out on eight dogs with Iarger wodnds gave comparabIe resuIts to those described in the guinea pig studies. I I. The ointment base proved to be entireIy satisfactory and we11 adapted for cIinica1 use. 12. A discussion of the cIinica1 possibiIities of chIorophyI1 in combination with the various antibacteria agents is presented, and its importance in the heaIing of chronic types of uIcerative Iesions is stressed. REFERENCES I. SMITH, L. W. and LIVINGSTON,A. E. ChIorophyII: an experimenta study of its water soIubIe deriva-

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tives in wound healing.

Smith,

Livingston-Chlorophyll

Am. J. Surg., 62: 35%

369, 1943. 2. SMITH, L. W. ChIorophylI: an experimenta study of its water solubie derivatives. I. remarks upon the history, chemistry, toxicity and antibacterial properties of water-soIuble chlorophylt derivatives as therapeutic agents. Am. J. Med. SC., 207; 647-654, 1944. 3. SMITH, L. W. and SANO, M. E. ChlorophyII: an experimental study of its water-soIubIe derivatives. IV. the effect of water-soluble chlorophyI1 and other agents upon the growth of fibroblasts in tissue cuIture. J. Lab. @z+Ch. Med., 29: 24146, 1944. 4. CLARK, A. M.,

GIBSON, T. and THOMPSON, M. L. PeniciIIin and propamidine in burns; eIimination of hemoIytic streptococci and staphyIococci. Lancet, I : 605, 1943. 5. FI.OREY, H. W. and CAIRNS, H. A preliminary report to the War Office and the Medical Research Council on Investigations concerning the use of peniciIIin in war wounds. Prepared in abstract for general pubbcation by L. P. Garrod. hit. Med. J., 2: 755, 1943; Lancer, 2: 742, 1943. 6. JEFFREY, J. S. Penicillin. &it. M. J., 2: 656, 1943; Lancet, 2: 639, 1943. 7. KEEFEK, C. S., BLAKE, F. G., MARSHALL, E. K., JK., I.OCKWOOO, J. S. and WOOD, W. B., JR. COLEBROOK, L.,

American Journalc,fSur~crq

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Penicillin in the treatment of infections; a report of 500 cases. J. A. M. A., 122: 1217, 1943. 8. LYONS, C. PeniciIIin therapy of surgicai infections in the U.S. army; a report. J. A. M. A., 123: 1007, 1943. 9. BUERGI, E. Porphyrins in the heaIing of wounds. J. A. &f. A., 121: 1237, 1943. to. BOEHRINGER,F. Wound treatment with chlorophyll ointment. Schweiz. med. Wcbnscbr., 72: 850, 1942; J. A. M. A., 121: 793, 1943.. I I. GRUSKI~, B. ChIorophyII, its therapeutic place in acute and suppurative disease. Am. J. Surg., 49: 49, 1940. 12. GAHAE, E., KLINE, P. R. and FINKLE, T. H. ChIorophyII in the treatment of uIcers. Arch. Dermat. P Sypbil., 47: 849-85 I, 1942. 13. WALLACE, G. I. and MOORMAN, H. E. Bacteriology of feet and incidence of dermatophytosis of feet of high school students. Arch. Dermat. EYSypbil., 47: 816821, 1943. 14. GOLDBERG, S. L. The use of water soIubIe chIorophyI1 in oral sepsis. Am. J. Surg., 62: I 17-r 23, ‘943. 15. HOLIIES, G. W. and MUELLER, H. P. Treatment of post-irradiation erythema with chIorophyI1 ointment. Am. J. Roentgenol. @Y Rad. Therapy, 50: August, 1943.