Human skin contains prostagiandin synthetase (cyclo-oxygenase) (Greaves &
McDonald-Gibson, 1972), and certain prostaglandins are potent vasodilators in
human skin (Sondergaard & Greaves, 1971 ; Gryglewski, 1979).
It therefore became of especial interest to produce direct evidence of the role, if
any , of prostaglandins in ultraviolet erythemaI reactions in human skin. These
studies also afforded an opportunity to investigate the mode of interaction ofsteroid
and non-steroid anti -inflammatory drugs with the prostagiandin system in man .
The first step is to develop a techn ique for recovery of inflammatory exudate from
human skin . The use of suction bullae has proved a practical and quantitative
method (Black, Greaves, Hensb y, Plummer & Ead y, 1977). Briefly perspex suction
cup s are placed on lower abdominal skin and suction appl ied at 200 mm Hg below
atmospheric pressure. After about 90 min, bullae, 4 mm in diameter, develop
opposite perforations in a diaphragm sited within the cup. Exudate is obtained from
acetate at pH 3. After preliminary chromatographie separation, the products are
suitably derivatised and measured by quantitative gas liquid chromatography-mass
The time course of production of arachidonic acid (AA) , prostagiandin E2(PGE2),
prostagiandin F2U(PGF2U), prostagiandin 02(PG02) and prostacyclin (PGh) in skin
irradiated with UV -B (290-320nm) was studied (Black , Fincharn, Greaves &
Hensby, 1980; Black, Greaves & Hensby, in preparation). In six volunteer subjects
lower abdominal skin was irradiated with three times the minimal erythemal dose of
UV-B. The time course oferythema was assessed visually, and exudate was obtained
at 6 min, 6 h, 18 h, 24 hand 48 h after irradiation.
The changes in levels of AA and prostaglandins E2, F2U and 02 are given in
Table I. At 2 h after irradiation there was a significant rise in levels ofAA and PGE2.
At 24 h, levels of AA, PGE2, PGF2U and PG02 were all raised, compared with
Table1 The recoverable levels (mean ± s.e. mean) ofPGE2, PGF2U, PGD2 and arachidonic
acid measured by GLC-MS in exudates from control skin and at various time intervals after 3
minimal-erythema dosesofUV-B irradiation.
(ng mi-I) (ngml-I) (ngmi-I) (ng mi-I)
Control 276.3± 19.5 17.06±0.7 18.2± l.l 21.8± 1.3
Hours after 294.3±45.7 20.2± 1.8 ND 17.9±3.1
irradiation P>0 .7 P>O.I P>0 .2
2 488.0±83.2 23.9± 1.9 ND 26.1±5.7
6 360.2±69.3 28.2±3.3 ND 35.0±4.7
18 540±59.5 34.9±3.4 ND 34.9±6.7
24 785±56.8 49.4±5.2 32.4±2.9 34.3±4.8
P<O.OOI P<O.OOI P<O.OOI P<0.05
P valuesshowsignificance relative to contro!.
ND =Not done. n =number ofsampies.
Prostacyclin (PGh) con centrations (measured as the stable 6-keto Flu
metabolite by GLC-MS) were measured in exudate obtained from an additional
group of volunteer subjects at 0, 6 h, 24 h, and 48 h after irradiation . In un irradiated
UV INFLAMMATION AND ANTI-INFLAMMATORY DRUGS 417
skin the mean concentration of ö-keto PGFI a was 8.7 ± 0.9 ng mI-I (n = 11) rising to
a maximum of 43.0 ± 4.9 ng mI-I (n = 4) at 6 hand at 48 h the 6-keto PGF. a
concentration had returned to control (pre-irradiation) values (Figure 1). The
progressive increase in AA and PG values during the first 24 h after irradiation is
marked by a parallel increase in the intensity of erythema during this period, which
becomes maximal at 24 h. Of particular interest is the fact that erythema, although
having faded at 72 h is still maximally intense at 48 h although AA and PGE2 values
have returned to control pre-irradiation values. Taken as a whole, the results suggest
that prostaglandins may be mediators ofvascular events in the earlier stages ofUV-B
Figure 1 The recoverable levels (mean ± s.e. mean) of6-keto PGFla measured by GLC-MS
in exudates from control skin and at various intervals after UV-B irradiation. The numbers of
subjects at each time point are shown. P values show significance, relative to controllevels.
Biosynthesis of prostaglandins is inhibited by non-steroid anti-inflammatory
drugs, and the analgesie and antipyretic actions ofthis dass of drug are thought to be
attributable to this property (Ferreira, Moncada & Vane, 1971). It is therefore of
with corresponding changes in the blood supply of the irradiated skin (Black,
Greaves, Hensby, Plummer & Warin, 1978).
Five volunteer subjects were studied. Abdominal skin was irradiated as before with
three times the minimal erythemal dose of UV -B, and exudate was obtained by the
suction bullae technique at 24 h after irradiation. This procedure was carried out
before and immediately after treatment of the subjects with indomethacin 50 mg
eight hourly for 48 h. Erythema was graded visually, and blood flow measured by
photoelectric plethysmography, using the method of Ramsay & Challoner (1976).
The results are given in Tables 2 and 3. PGE2 and PGF2 (l levels were elevated in the
24 h UV -B reaction , but prior treatment with oral indometh acin totally inhibited
evoked increas e in PG activity. Arach idonic acid was increase d almost threcfold after
irradiation, but after indomethaci n the concentration was even higher (894 .0 ± 125.6
ng mI-I, compared with 785.4 ± 56 .8 ng mi- I before indometh acin) . However th is
ditTerence did not reach statistical signi ficance. These results therefore provide in vivo
evidence in man to support th e view th at non-stero id anti-inflarnma tory drugs
suppress prostagiandin biosynthesis. The pharmacological fmdi ngs in non-irradiated
skin were also of interest (Table 2). Indomethacin ca used a mo re than twofold
Table 2 The recoverable levels (mean ± s.e. mean) of PGE2, PGF2(l and arachidonic acid
determined by GLC-MS in control and UV-B irradiated skin at 24 h, before and after
Before indomethacin Before indomethacin
PGE2 21.9± 1.2 15.7±2.9 49.4±5.2 16.6± 1.3
PGF2a 18.2± 1.1 15.2±4.5 32.4±2.9 14.0± 1.4
Arachidonic 284 .6±24.6 723.0±74.0 785±56.8 894.0± 125.6
(ng ml'") P <O.OOI P <O.OOI P <O.OOI
P values show significancerelative to control levels.
visually and by photoelectric ple th ysmography showed a six to sevenfold increase
24 h after irradiation, which correlated weil with the rise in AA and PG activity.
However after indomethacin , blood flow was suppressed by about one-quarter to
one-third, although PGE2 and PGF2a levels were completely suppressed to control
values. This suggests that prostaglandins may not alone account for the intense
Table 3 Visual erythema grade and total blood flow determined by photoelectric
plethysmography in controI and UV-B irradiated human skin before and after oral
*Erythema scale: 0 =no erythema; I=minimal erythema; 2=moderate erythema; 3=deep red
P values showssignificancerelative to 24 h UV-irradiated levels.
UV INFLAMMATION AND ANTI-INFLAMMATORY DRUGS 419
It is instructive to compare these fmdings with the response of evoked PG activity
in human skin to systemic corticosteroids. For this study, the fact has been used, that
topical application of an ester of nicotinic acid, called tetrahydrofurfuryl pyridine-3-
carboxylate cream (Trafuril Cream, Ciba), causes a sustained inflammatory response
after a latent period of 10-15 min associated with increased prostagIandin activity in
the inflammed skin (Plummer, Hensby, Black & Greaves, 1977). Trafuril Cream
(1 ml volume) was appliedto 120 cm- area of the lower abdominal skin of six
volunteer subjects. Soon after the appearance of erythema, exudate was obtained
from the inflammed skin by the suction bullae technique. Exudate was also obtained
from adjacent control sites to which only the base of the Trafuril Cream had been
applied. These procedures were carried out before and after administration of
prednisolone, I g orally daily for two days in six subjects. These were patients
receiving pulsed doses ofsteroids for therapy ofextensive alopecia areata.
Table 4 The effect of prednisolone on Trafuril-induced prostaglandin recoverable levels
(ngmI-I) in control and Trafuril treated skin. Resultsare shownas mean ± s.e. mean.
acid 246.9±25.3 212.7±37.3 500.3±60.6 239.2±56.2
The results summarised in Table 4 show that Trafuril application to skin evokes
19.2 ± 3.4 ng mi-I, but after Trafuril it rose to 37.3 ± 2.0 ng mI-I. However prior
administration of prednisolone was followed by a considerable reduction in the
expected rise following Trafuril application (26.1 ± 2.3 ng mi-I). Similar results were
obtained with PGF2a. The arachidonic acid results were of especial interest. The
mean level of arachidonic acid rose from 246 .9 ± 25.3 ng mI-I, in skin prior to
Trafuril application, to 500 .3 ± 60.6 ng mI-I, after Trafuril application. However
prior administration ofprednisolone totally inhibited this evoked rise in arachidonic
acid, the mean post-steroid value being 239 .2 ± 56.2 ng mI-I. These results contrast
with those obtained using indomethacin on UV -B - evoked increase in prostagIandin
activity (see above) where indomethacin, although causing a similar suppression of
PG activity, caused a further increase in arachidonic acid levels above those obtained
in UV -irradiated skin without indomethacin. Thus unlike indomethacin, steroids
appear to inhibit biosynthesis ofprostaglandin by preventing release or formation of
arachidonic acid. Possible mechanisms include inhibition ofrelease ofthe Iysosomal
enzyme, phospholipase A2, as proposed by Weissman (1969). Recent work (Danon &
formation ofa protein inhibitor of phospholipase A2.
Two other fmdings in the Trafuril-prednisolone study are also of importance.
Although prednisolone caused a substantial reduction in the expected increase in
of inflammation may be involved in the Trafuril reaction. Finally the results of
Table 4 also show that prednisolone had little or no elTecton arachidonic acid levels
in healthy skin (no Trafuril). This suggests that the source of arachidonic acid in
normal skin (which is not steroid-sensitive) dilTers from the source of the elevated
levels in inflammed skin (which is apparently steroid-sensitive).
In conclusion, it is clear that ultraviolet irradiation, as weil as other types ofinjury,
evoke increase in levels of pro-inflammatory prostaglandins in human skin.
Although these prostaglandins may weil playa part in the observed inflammatory
activity, but they act at different sites. Steroids act at early stage, by suppressing
formation ofthe prostagiandin precursor arachidonic acid. In contrast, indomethacin
suppresses the cyclo-oxygenase catalysed conversion of arachidonic acid to prostaglandin.
Black, A. K., Fineharn. N., Greaves, M. W. & Hensby, C. N. (1980). Time course ofarachidonic
acid, prostagiandin D2, E2 and Fw levels in human skin following ultraviolet B (UV-B)
irradiation (290-320 nm). Brit. J. c1in. Pharmac.. in press.
Black, A. K., Greaves , M. W., Hensby, C. N., Plummer, N. A. & Eady, R. A. J. (1977). A new
method for recovery of exudates from normal and inflammed human skin. Clin. exp.
Black, A. K., Greaves, M. W., Hensby, C. N., Plummer, N. A. & Warin, A. P. (1978). The effects
ofindomethacin on arachidonic acid and prostaglandins E2 and F2a levels in human skin
24 h after UV-B and UV-C irradiation. Brit. J. clin.Pharmac.. 6,261-266.
Danon, A. & Assouline, G. (1978). Inhibition of prostaglandin synthesis by corticosteroids
requires RNA and protein synthesis . Nature, 273, 552-554.
Eaglestein, W. H. & Marsico, A. R. (1975). Dichotomy in response to indomethacin in UV-C
and UV-B-induced ultraviolet light inflammation. J. invest. Derm..65,238-240.
Goldsmith, P., Tuck, A. F., Foot, J. S., Simmons, E. L. & Newson , R. L. (1973). Nitrogen oxide,
nuclear weapon testing, Concorde, and stratospheric ozone. Nature. 244,545-551.
Greaves, M. W. & McDonald-Gibson, W. (1972). Inhibition of prostaglandin biosynthesis by
corticosteroids. Brit. med. J., 1, 83-84.
Gryglewski , R. J. (1979). Prostacyclin as circulatory hormone. Biochem. Pharmac.. 28,
Gschnait, F. & Pehamberger, H. (1977). Indomethacin does not affect PUV A-induced
erythema. Arch. Derm. Res.. 259 ,109-111.
Omdahl, J. C. & de Luca , H. F. (1973). Regulation of vitamin D metabolism and function.
Parrish, J. A., Fitzpatrick, T. B., Tanenbaum, L. & Pathak, M. B. (1974). Photochemotherapy of
psoriasis with oral methoxalen and long wavelength ultraviolet light. New Eng. J. Med.,
Plummer, N. A., Hensby , C. N., Black, A. K. & Greaves, M. W. (1977). ProstagIandin activity in
sustained inflammation ofhuman skin, before and after aspirin. Clin. Sei. mol. Med.. 52,
Ramsay, C. A. & Challoner, A. V. J. (1976). Vascular changes in human skin after ultraviolet
irradiation. Brit. J. Derm.. 94,487-493.
Sondergaard, 1. & Greaves, M. W. (1971). Prostaglandin E: effect on human cutaneous
vasculature and skin histamine. Brit. J. Derm.. 84,424-428.
Weissman, G. (1969). The effect of steroid drugs on lysosomes. In Frontiers 0/ Biology, pp.
276-295. Amsterdam: North Holland PublishingCo.
S.SHUSTER, M. RAWLINS, S. ROGERS, B. CHADKlRK,
University Department ofDermatology.
Newcastle-upon- Tyne NE14LP, England
compared the dithranol regimen as developed by Ingram (1954) with the more recent
treatment with 8-methoxypsoralen and ultraviolet Airradiation (PUVA).
All patients had chronic plaque psoriasis and 19 were treated with dithranol using the
Ingram (1954) regimen and 27 with PUVA as previously described (Rogers, Marks,
Vella Briffa, Warin, Shuster & Greaves, 1979). Measurements were made before and
throughout the treatment on plaque on the forearm and adjacent clinically unaffected
skin . The following measurements were made:
2. Friction The coefficient ofdynamic friction, u, was measured with the Newcastle
Skin Friction Meter (Comaish, Harborow & Hofmann, 1973).
3, Plaque thickness Plaque thickness was measured with Harpenden calipers from
which one spring had been removed to reduce tissue compression as described by
I. Temperature The mean ± s.e. mean pre-treatrnent temperature of uninvolved
skin was 32.5 ±O.l'C and plaque temperature was 32.2 ±O.l'c. This small difference
422 S. SHUSTER, M. RAWLlNS, S. ROG ERS, B.CHAD KIRK,J. MARKS & J. S. CO MAISH
was significant (P<0.02) but quite inadequate to serve as a measure ofimprovement
in individuals. Furthermore the considerable variation in plaque temperature during
treatment was often greater than that between plaque and uninvolved skin .
2. Friction Before treatment the mean ± s.e. mean coefficient of dynamic friction of
uninvolved psoriatic skin (0 .34±0 .03~) was similar to that of normal subjects but
significantly less than in the plaques (0.2 ± .02~) Surprisingly friction was not
altered significantly at the end of a course of treatment wish dithranol or PUV A
(0.25 ± 0.02~, P <0.1) although c1inically the psoriasis had responded and the skin
surface appeared normal. Consequently the coefficient of dynamic friction was not
suitable for monitoringresponse ofpsoriasis to treatment.
3. Plaque thickness The mean ± s.e. mean plaque thickness before treatment was
0.6 ± 0.08mm and it decreased progressively with treatment although the response
was different in dithranol and PUV A treated patients.
PUVA Plaque thickness decreased log-linearly with time and with a half-time (T'I,)
of 12 days. Clearing was related to initial plaque thickness, and the tim e taken for
an individual plaque to c1ear correlated weil with its initial thickness (r = 0.77).
Dithranol As with PUV A plaque thickness decreased log-linearly with time but it
was faster, T'!, being 9 days. Also in contrast with PUV A there was no correlation
between init ial plaque thickness and time to clearing ofthe rash (r =-0.23).
4. Water loss Thc mean ± s.e. mean water loss from psoriatic plaque before
treatment was 26.9±4.12g-2 h-I compared with 1O.0±I.5g-2 h-I from adjacent
uninvolved skin. There was an initial lag in response after which water loss fell
with treatment unt il at the point when the rash had gone it had reached levels
similar to those in c1inically uninvolved skin in the PUV A treated patients. In the
dithranol treated patients however water loss was still increased when the rash had
c1eared. There was a correlation between initial plaque thickness and water loss
which was sigmoid for PUV A and curvilinear with a wide scatter for dithranol.
Of the four characteristics of psoriatic plaques measured two were unsatisfactory
for following the responses to treatment, and for different reasons . Although skin
surface temperature was consistently lower in the untreated plaques than in nearby
non -lesional skin the difference was small. Large fluctuations in skin temperature
occurred both above and below the pretreatment figures which were probably due
to the variable inflammatory response to the treatment. For these reason s skin
temperature is unlikely to serve as a satisfactory basis for monitoring response to a
The coefficient of dynamic friction was consistently decreased in the lesions as
compared with the c1inically normal skin where it was no different from that in the
skin of normal subjects. The reason for this considerable change in dynamic
friction is not c1ear nor is it obvious why it persisted in what appeared to be
completely resolved plaques. This persistence does, however, preclude the use of
skin friction for the measurement of response of the rash to treatment. By contrast
with skin temperature and friction , skin thickness and transepidermal water loss
were satisfactory measures of c1inical response as both showed similar incremental
improvement with time. The measurement of both was equally simple although
the apparatus for the latter was sophisticated. Transepidermal water loss showed
greater variation than did skin thickness and it corresponded less with c1inical
appearance both during the first week oftreatment and at its termination.
Both measurements showed the different response to PUV A and dithranol and
the slope of the tirne-response curves should serve as a useful basis for
comparison of different treatments. In comparing the response to dithranol and
PUVA AN D DITHRA NOL IN PSORIASIS 423
PUVA in this wa y an important difference was apparent. With dithranol the rate
of improvement (slope of improvement curve) was unrelated to initial plaque
thickness so that the time taken for the plaques to clear was constant regardless of
initial thickness. With PUVA by contrast the rate of improvement was related to
initial plaque thickness so that the time to clinical clearance could be predicted
irradiation has a limited penetration and its effect is likely to be restricted by
This work was suppo rted by an MRC Programme Grant.
Comaish, J. S., Harborow, P. R. H. & Hofman, D. A. (1973). A hard-held friction meter. Brit.
Cook, L. J. & Shuster, S. (1980). Histamine weal formation and absorption in man. Brit. J.
Ingram,J. T. (1954). The significance and management ofpsoriasis. Brit. med. J.. 2,823 .
Rogers, S., Marks,J., Vella Briffa, D., Warin, A., Shuster, S. & Greaves, M. (1979). Comparison
of photochemotherapy and dithranol in the treatment of chron ic plaque psoriasis. Lan cet,
Institute ofMicrobial Chem istry,
M echanisms oftherapeutic action
The structures of bleomycin s, including their stereochemistry as shown in Figure I,
were conclusively determined by a study in the author's institute in 1978 (Takita,
Muraoka , Na kata ni, Fujii, U mezawa, Naganaw a & Umezawa, 1978b). The
shown in Figure I ha s been supported by cr ystal X-ra y ana lysis and also by the
chemical synthesis of pyrimidoblamic acid. Moreover , th is structure has been
confirmed by 15N-n. m.r. of bleomycin A2 (Naganawa , Takita, U mezawa & Hull,
1979) and also by ma ss spectroscopic anal ysis ofbleomycins BI' wh ich gave the exact
mol ecular weight of bleomycin (Macfarlane, Fujii, Takita & U meza wa, 1980). The
main structural group co mmon to a ll bleomycin s is called bleomycinic acid (Figure
termina l amine . As shown in Figure 1, variou s bleomycin s which are different from
on e another in the terminal amine are produced by a bleornycin-producing strai n.
The presen t bleomycin used c1inically consists mainIy of bleomycins A2 an d B2.
Ind ividual bleomycins shown in Figure 1, had a Iower therapeutic index against
Ehrlich carcinoma than the mixt ure and therefore , the mixture containing A2 and
B2 in th e ratio of about 3:1 as th e main com ponents was introduced into the c1inic
In the study of th e th erap eutic action of bleomycin aga inst sq uamo us cell
ca rcino ma, it was found that various anima l cell s conta ined an enz yme which
inactiva ted bleomycin (U mezawa, Ho ri, Sawa , Yoshioka & Takeuchi, 1974;
Umeza wa , Ish izuka, Ao yagi & Tak euchi, 1976). This enz ym e was named bleomycin
hydrolase. It hydrol yzes the a-aminocarboxamide bond of the pyrimidoblamyl
moiet y of bleo myci n. The content ofthis enzyme is signi ficantly lower in squamous
ca rcinoma induced by 3-methylcholanthrene in mouse skin than in sa rcoma induced
by the same carcinogenic agent, and the former is sensitive to bleomycin treatment
but the latter is resistant. A low content ofbleomycin hydrolase is one ofthe reasons
why squamous cell carcinoma is susceptible to bleomycin treatment (Umezawa,
° HOü~i jJ 2 ' 'f ° * * NH N S
CH! N~+ 0 + I H * N CH! 0 CH S + H H!
Demelhyl-A2 (R=NH-(CH2h-S-CH!1, A2 (R=NH-(CH.h-S, 1, + CH!
A2/-a (R=NH-(CH21.-NH2), A2/-b (R=NH-(CH2h-NH2),
A2/-c (R=NH-(CH21.-Cß1, A5 (R=NH-(CH2h-NH-(CH2).-NH.),
AG (R=NH-(CH2h-NH-(CH2).-NH-(CH2h-NH2), BI' (R=NH.),
B2 (R=NH-(CH2).-NH-C-NH.), B4 (R=NH-(CH.).-NH-C-NH-(CH2).-NH-C-NH.).
Pepleomyci n: R=NH-(CH2h-NH-9-D I~
BAPP: NH- (CH2h-NH-(CH2h-NH-(CH2h-CH!
Figure1. Structureofnatural bleomycins, pepleomycin, BAPPand bleomycinic acid.
Bleomycin binds strongly with cupric ions and forms an equimolar complex. This
bleomycin-copper complex is resistant to bleomycin hydrolase and is not inactivated
by this enzyme. The structure of bleomycin -copper complex has also been proposed
(Takita, Muraoka, Nakatani, Fujii , Iitaka & Umezawa, 1978a; Umezawa & Takita,
1980) and this structure was supported by mass spectroscopic analysis ofbleomycin
BI' (Macfarlane et al., 1980). Bleomycin-copper complex binds to DNA: the
bithiazole moiety binds to the guanine moiety ofDNA and the terminal amine is also
involved in the binding (Kasai, Naganawa, Takita & Umezawa, 1978). Although
bleomycin-copper complex binds to DNA, it does not cause DNA fragmentation.
RECENT STUDIES ON BLEOMYCIN 429
The binding of cupric ions to bleomycin is very strong. But, if the cupric ion of the
bleomycin-copper complex is reduced in the presence of a cuprous ion-chelating
agent such as neocuproine, then the cuprous ion thus formed is transferred to
neocuproine and copper-free bleomycin is produced (Takahashi, Yoshioka, Matsuda
On the basis of the structure of bleomycin-copper complex, the structure of
bleomycin-ferrous complex, as shown in Figure 2, was proposed (Takita et al..
1978a). This structure is being confirmed by n.m.r. and e.s.r. studies of ferrous
complexes of bleomycin, deglycobleomycin (the peptide part of bleomycin) by
Sugiura at the Faculty of Pharmaceutical Sciences, Kyoto University, and
Morishima, at the Faculty of Engineering, Kyoto University. The structure of
bleomycin-ferrous complex proposed by Oppenheimer, Rodriguez & Hecht (1979)
does not agree in various points with the n.m .r ., e.s.r. and mass spectroscopic
analyses offerrous complexes ofbleomycin, deglycobleomycin and so on .
"'" H N,----rf CO-terminal amine
H N,~,~~J;\J/tH.1p-~Thr-NH-JtsjJ
AHMP: (2S.3S,4R)-4-amll1o-3-hydroxy-2-methylpentanolc acid
Figure 2. Bleomycin- Fert-O, complex.
amine moiety binds also to DNA. The oxygen molecule which binds to the ferrous
ion ofthe bleomycin-ferrous complex binding to DNA is activated and this activated
oxygen molecule, or a radical produced, reacts with the deoxyribose moiety ofDNA,
and as a result of this reaction, DNA fragmentation occurs. After the reaction,
bleomycin remains intact (Ekimoto, Kuramochi , Takahashi, Matsuda & Umezawa,
1980). Thus, the type of the reaction of bleomycin with DNA is a kind of enzymic
reaction. As first noticed by Müller & Zahn (1976), in this point, bleomycin is a
On the basis of the chemistry of bleomycin as described above, the behaviour of
bleomycin in vivo has been elucidated as follows (Umezawa, 1976): injected
bleomycin binds with cupric ions in the blood and a bleomycin-copper complex is
produced. After penetration into the cells, the cupric ion of the bleomycin-copper
complex is reduced by intracellular reducing agents, for example, cysteine, and the
cuprous ion thus formed is transferred to a cellular protein which can selectively bind
with cuprous ions. This protein was first discovered by the study ofbleomycin action
(Takahashi et al.. 1977). Copper-free bleomycin thus formed undergoes hydrolysis by
bleomycin hydrolase. Some copper-free bleomycin which escaped from the action of
bleomycin hydrolase reaches the nuclei and binds with DNA. The oxygen molecule
binds to the ferrous ion of bleornycin-ferrous complex binding to DNA and DNA is
As already described, the therapeutic action of bleomycin against squamous cell
carcinoma has been shown to be due to a low content of bleomycin hydrolase and a
high concentration of bleomycin in this tumour (Urnezawa, 1976). Hodgkin's
lymphoma is more sensitive to bleomycin treatment than squamous cell carcinoma.
If there are cancer cells which lack a system to repair the damage caused by
bleomycin, such cancer cells should be most sensitive to bleomycin treatment. In this
connection, it is interesting that even a very small dose (0.5 or 1.0 g daily) can show
the effect ofdecreasing this tumour (Yagoda & Krakoff, 1976). The mechanism ofthe
therapeutic action against Hodgkin's lymphoma should be different from that against
DeveIopmentof effectivederivativesandanaIogues
It is known that the complexes of bleomycin with radioactive metals such as 57C O,
1I1 In, 99mTc are preferentially taken up by cancer cells and are useful for diagnosis of
cancer (Nouel, 1976). This indicates the selective uptake ofbleomycin by cancer cells
compared with normal cells including those involved in inflammation and suggests a
possibility of fmding new bleomycins which have a wide anticancer spectrum. Such
bleomycins may be found among bleomycins resistant to bleomycin hydrolase.
As already described, the a -aminocarboxamide bond in the bleomycin molecule
is hydrolyzed by bleomycin hydrolase. Therefore, modiftcation of this molecular
part gives derivatives resistant to bleomycin hydrolase. The N-a-methyl derivative
has been conftrmed to be resistant to bleomycin hydrolase (Fukuoka, Muraoka,
Fujii, Naganawa, Takita & Umezawa, 1980). However, the therapeutic index ofthis
derivative against Ehrlich carcinoma was signiftcantly smaller than the present
bleomycin (A2 and 82). Further study has indicated the presence of derivatives
which are resistant to bleomycin hydrolase and have a high therapeutic index against
Ehrlich carcinoma. It is possible to develop new bleomycins which have a wide anticancer spectrum.
As discussed earlier, the various bleomycins produced by fermentation were
different from one another in the terminal amine moiety, as shown in Figure I. This
suggested that an amine added to the fermentation media would be incorporated into
the bleomycin molecule. In fact, such a bleomycin was produced and the addition of
the amine suppressed the production ofother bleomycins. By this method, more than
200 artificial bleomycins, which have different terminal amines, have been prepared.
Moreover, an enzymic method to prepare bleomycinic acid from bleomycin B2 and a
chemical method to prepare bleomycinic acid from bleomycin demethyl-A2 were
established and various bleomycins containing different terminal amines have been
prepared also by chemical derivation ofbleomycinic acid (Urnezawa, 1976). These
various bleomycins differ in the degree of their toxicity. Bleomycins containing
Table l. Pulmonary toxicity ofnew bleomycins. Bleomycin= RCOR'
'All are ellective in treatment 01 squamous cel l carcinama in
mouse skin induced by methylcholanthrene.
RECENT STUDlES ON BLEOMYCIN 431
strong basic groups, such as bleomycin B4 and B6 wh ich have two or more guanidine
groups, have a strong renal tox icity. Bleomycin M5196 (Table I) wh ich contained an
amidine group also has a strong renal toxicity.
A method testing lung toxicity in mice was established by Matsuda, Yoshioka,
Yamashita, Ebihara, Umezawa, Miura, Katayama, Yokoyama & Nagai (l978b).
index was 0.25 , taking the index ofthe present bleomycin as 1.0. Its lower pulmonary
toxicity was also confirmed by the pulmonary function test after administration to
cancer patients. Thus, the result ofthe pulmonary toxicity test in mice was shown to
be useful in predicting the pulmonary toxicity to cancer patients. Applying this test ,
bleomycin PEP and BAPP were found to have a low pulmonary toxicity and selected
as those worthy of c1inical testing. Moreover, these bleomycins showed strong
inhibitory activity towards squamous cell carcinoma, induced in mouse skin by
methylcholanthrene as weil as the present bleomycin (A2 and B2). A therapeutic
effect on rat stomach carcinoma induced by N-methyl-N-nitro-N'-nitrosoguanidine
was also shown (Matsuda, Yoshioka , Takahashi, Yamashita, Ebihara, Ekimoto,
Abe, Hashimoto & Umezawa, 1978a) . The present bleomycin (A2 and B2) was not
efTective against this stomach carcinoma. This effect of PEP was suggested to be
stronger than BAPP. Thus, bleomycin PEP, which was named pepleomycin, was
examined further in detail. Pepleomycin showed astronger inhibition of lumbar
lymph node metastases of rat hepatoma AH66 cells, inoculated to testis compared
with the present bleomycin (A2 and B2) (Matsuda et al., 19'i8a).
The effects 01' pepleornycin on experimental animal tumours described above and
found by the tests of the National Cancer Institute, USA indicate that pepleomycin
and BAPP have a wider antitumour spectrum against experimental animal tumours.
Pepleomycin has been c1inically studied during the last four years. The frequency
of pulmonary side efTects caused by pepleomycin during the treatment of carcinoma
of the skin, head and neck was suggested to be lower than that caused by bleomycin
(A2 and B2). Moreover, the degree ofactivity ofpepleomycin in decreasing the size of
tumours was suggested to be stronger than bleomycin (A2 and B2). As shown in
Figure 3, thc regression of carcinoma in the head and neck became apparent after the
administration of about 58 mg of pepleomycin in total, as the mean. This indicates
Figure 3. Regression ofhead and neck tumours in patients treated with either pepleomycin (e)
or bleomycin (0). More than 90% regression ofthe tumour is indicated by +++. The number of
that the etTect ofpepleomycin appears more quickly than that ofbleomycin (A2 and
82). In the caseofbleomycin (A2 and 82), tumour regression became apparent after
Pepleomycin has been proved to have a wider anticancer spectrum against human
ditTerentiated prostatic cancer in combination with radiation has been emphasized.
Moreover, adenocarcinoma cystica, a malignant tumour ofthe mouth, was resistant
to bleomycin, but all five cases tested responded to pepleomycin treatment (Inuyama,
1979, personal communication). About eight years ago, a small-scale clinical study of
bleomycin A5 suggested that this bleomycin might be etTective against breast cancer.
Clinical study has indicated that pepleomycin would be useful in the treatment of
As shown by the study of pepleomycin, a change of the terminal amine and a
modification ofthe a-aminocarboxamide moiety ofbleomycin can give derivatives
which have a lower toxicity and wider anticancer spectrum than bleomycin (A2 and
82). The study ofderivatives and analogues will be continued to develop more useful
Combination of bleomycin with other treatment modes
The combination ofbleomycin with other treatment modes has increased the rate of
organ (Edsmyr, 1976). It is weil known that combination of bleomycin with a vinca
alkaloid and cis-platinum has increased the rate of complete regression of testis
Additional treatment with an immune-enhancing agent has been suggested to
augment the therapeutic etTect ofbleomycin. For example, additional treatment with
bestatin, a small molecular immunity enhancing agent (Umezawa et al.. 1976;
Umezawa, 1979), increases the antitumour etTect of bleomycin. If Ixl0 6 cells of an
Ehrlich carcinoma are inoculated into a mouse footpad , the growth ofthis tumour is
resistant to treatment with bleomycin or other antitumour compounds. Neither the
daily administration of bestatin alone nor the daily administration of 100 ~g of
bleomycin alone for six days showed any inhibition of the growth of this tumour.
However, addition of 100, 10 or 1.0 bestatin daily to bleomycin treatment, as
shown in Table 2, caused a strong inhibition ofthe tumour. The addition of 1,000
Table 2. Effectofbestatin enhancing the antitumour effectofbleomycin on Ehrlich carcinoma
cells inoculated into mouse footpads. Bleomycin was given intraperitoneally at a dose of 1001t1
daily for six days, from one day after inoculation of I x 106 tumour cells. Bestatin was given
orally for six days from one day after the inoculation of the tumour cells. The size of each
tumour was measured 30 days after inoculation. The standard deviation did not exceed 15%.
The mean sizeofthe control without treatment was48 .9.
RECENT STUDIES ON BLEOMYCI N 433
bestatin did not enhance the efTect of bleomycin. As has been reported, thi s high
dose of bestatin does not enhance delayed-type hypersen sitivity to sheep red blood
cell s (Umezawa et al., 19 76).
Bestatin treatment al so enhanced the effect of bl eomycin on rat hepatoma AH66.
This hepatoma was ma intained as its asciti c form and IxlO7 cells, obtained from the
ascites form, were inoculated subcutaneously in Donryu rat s. The rats were treated
with be statin (0.5mg kg-I dail y) alone, bleomycin (0.5mg kg-I da ily) alone, or bestatin
plus bleomycin daily, from one day aft er the inoculation for 10 da ys. The weight of
each tumour was measured fourteen day s after ino cul ation. Then, the weights of
tumour were as folIows: the control without an y treatment, 7.66 ± 0.80 g; bestatin
alone, 5.03 ± 0.51 g (34 .3% in hibition); bleomycin a lone , 4.72 ± 0.43 g (38.4%
inhibition); bestatin plus bleomycin, 2.09 ± 0.51 g (72 .7% inhibition).
Bleomycin ha s been known to be not immunosuppressive . On the other hand, it is
known th at supp ressor cells increase in mice bearing a tumou r. IMC carcinoma is a
mouse carcinoma which appeared spontaneously in CDFI mic e in the author's
week s after subcutaneous inoculation of 106 IM C ca rcinoma cells, promoted the
growth of thi s tumour inoculated into a footpad. The cells taken from mice two
weeks aft er the inoculation of 106 IMC carcinoma cells were a mixt ure ofefTector and
suppressor cell s. The author ass umed that there wer e antitumour substa nces wh ich
attacked suppressor cell s preferentially co m pared with efTector cell s. In fact, in our
study, suppressor cells have been suggested to be more sensitive to th e cytotoxic
action of aclacinomycin than efTector cell s, Suppressor cell s in th e mixture of
pepleomycin than to that of bleomycin . In the experiment sho wn in Table 3,
cell s and cultured with pepleomycin (1.0 or 0.1 lJgml-l) and bleomycin (1.0 or 0.1
ugml") for 60 min a nd washed. These lymphoid cell s were cultured with IMC
carcinoma cell s in th e ratio of 10:1 (2.5 x 106 lymphoid cells and 2.5 x 105 IMC
measured 14 days thereafter. As shown in Table 3, the lymphoid cell s obtained from
normal mice did not give an y in fluence on the tumour growth. The treatment ofthese
cell s with bleomycin o r pepleomycin produced no influence. Intravenous inj ection of
lymphoid cells obtained from mice two weeks a fter the inoculat ion of tumour cells
showed no significant influence on the tumour growth but the intravenous inje ction
Table 3. The influence of treatment with pepleomycin (PEP) and bleomycin (BLM) on the
anticancer effect of lymphoid cells of normal mice, or of mice two weeks after inoculation of
of pepleomycin-treated lymphoid cell s of IMC-carcinoma-bearing mice produced a
strong inhibition on the growth of the tumour. This suggests that suppressor cells
were affected by pepleomycin preferentially compared with effector cclls which were
produced as the immune response to the tumour cells inoculated. This effect of
pepleomycin was st ro nge r than that ofbleomycin (Table 3).
It is becoming interesting to select bleomycin derivatives which attack suppressor
cells more selectively than the present bleomycin (A2 and 82).
Mechanisms of therapeutic action of bleomycin have been studied in relation to its
chemical structure, and derivatives and analogues have been studied in detail. As
already shown by the study of its derivatives, it is possible to develop derivatives and
spectrum. This study is being continued and will contribute to the increase in the rate
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HIGH DOSE-POTENCY IN LEUKAEMIA
B. F. CAIN, B. C. BAGULEY, W. A. DENNY &
Ca ncer Chemo therapy Research Laboratory.
Universitv of Auck land School ofMedicine. . . New Z ealand .
From among a large number of acridine deri vatives screened for antitumour acti vity,
m-AMSA (Scheme I; Figure I), has been found to pro vide excellent tumour
inhibition in a range ofmodel an ima! systems (Cain & Atwell, 1974 ). The high, broad
spectrum, experimental acti vity of this compound has prompted clinical trials and
existing indications are that this agent has clinica! activity again st certain categories
ofcancer (Von HofT, Howser, Gormley, Bender , G!aubiger, Levine & Young, 1978;
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