Arch. Derm. Res. 254, 87--93 (1975) © by Springer-Verlag 1975

D-Penicillamine in Dermatology: Influence on Enzymatic Activities of Human Skin in vitro

W. Raab and B. Gmeiner

University Medical School, Department of BIedical Chemistry (Chairman: E. Kaiser, N[. D.), Vienna (Austria)

Received May 16, 1975

Summary. By in vitro assay, 6 important enzymatic activities of human skin homogenates were determined following an incubation with D-penicillamine in concentrations between 10 -4 and 10 mg/ml, i.e. 67)< I0 -s and 67 raM/1. The following enzymatic activities were re- corded: lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), glyceraldehyde-3-phosphate dehydrogenase {GAPDH), alkaline phosphatase (AP), acid phosphatase (AcP), and "leucine aminopeptidase" {LAP). A dose-dependent activation by D-penicillamine occurred in the case of G-6-PDH- and AcP-activities, a dose-dependent in- hibition by D-penieillamine was found with AP- and GAPDH-activities. LDtt- and LAP- activities remained unchanged in the presence of D-penicillamine in concentrations up to 10 mg/ml (67 raM/l). From the data of pharmacokinetic studies in rats it may be concluded that concentrations of D-penicitlamine which influence enzymatic activities may easily be reached in vivo, under the conditions of treating rheumatoid arthritis and Morbus Wilson. The biochemical actions of D-penicillamine are briefly discussed with special regard to dermatolo- gical therapy and dermatological unwanted side-effects.

Zusammen/assung. In vitro wurden 6 wichtige Enzymaktivit~ten menschlicher Haut nach Inkubation mit D-Penicillamin in Konzentrationen zwischen 10 -4 und 10 mg]ml (67 × 10 -5 und 67 talK/l) bestimmt. Folgende Enzymaktivit~ten wurden untersucht: Lactatdehydroge- nase (LDH), Glueose-6-phosphat Dehydrogenase (G-6-PDH), Glycerinaldehyd-3-phosphat Dehydrogenase (GAPDH), alkalische Phosphatase (AP), saure Phosphatase (AcP) und ,,Leu- cinaminopeptidase" (LAP). Eine konzentrationsabh~ngige Aktivierung durch D-Penicill- amin wurde bei der G-6-PDH- und bei der AeP-Aktivit~t festgestellt; eine konzentrations- abh~ingige Hemmung fand sich bei der AP- und GAPDH-Aktivit~it. Die LDH- und LAP- Aktivit~ten blieben unbeeinfluBt, zumindest nach Inkubation mit D-Penicillamin in Konzen- trationen bis 10 mg/ml (67 mM]l). Aus den Ergebnissen pharmakokinetischer Untersuchungs- serien an Ratten l~l~t sich sehliel]en, dab bei der D-Penicillamin-Behandlung yon rheuma- toider Arthritis oder yon Morbus Wilson in der Haut der Patienten Konzentrationen erreicht werden kSnnen, die Enzymver~nderungen bewirken. Abschliel3end werden die bioehemischen Wirkungen yon D-Penicillamin, die zu Enzymaktivit~tsver~nderungen fiihren kSnnen, kurz diskutiert; speziell eingegangen wird anf die Belange der dermatologischen Therapie und auf die unerwiinschten Wirkungen des D-Penieillamins an der Haut.

Introduction

I n those last years, D-peniei l lamine (fl, fl ' -dimethylcysteine) is being used no t only for the t r ea tmen t of Morbus Wilson, heavy metal intoxicat ions, and cyst inuria bu t for rheumato id arthri t is , a far more common disease, too. The interest in this drug is shifted from its chelating properties to its other biochemical activities. Some of the mechanist ic bases of the various therapeutic actions of D-penicill- amine are still no t fully understood. D-Penici l lamine exerts three ma in activities

88 W. Raab and B. Gmeiner

[25] : i t possesses chelating act ivi ty, it produces exchange reactions with disulfides, and it reacts with aldehyde groups.

Trials are being performed as to the therapeut ic use of D-penici l lamine in col- lagen diseases and in condit ions of d is turbed kera t in iza t ion [2, 6, 9,11,13, 21,40, 41]. Wi th these indications, D-penici l lamine is receiving higher a t t en t ion from dermatologists. Pharmacokinet ic studies [24,33] revealed the fact t ha t large amount s of D-penici l lamine are deposited in the skin ("dermotropic" proper ty of D-penieil lamine).

All 3 types of biochemical act ions of D-penici l lamine m a y provoke changes in enzymat ic act ivi t ies: ac t iva t ion or inhib i t ion ma y occur. The observat ion of relat ively high concentra t ions of D-penici l lamine in the skin suggested to in- vest igate the changes in some impor t an t enzymes of h u m a n skin following an incuba t ion with D-penici l lamine. The results of such studies in vitro will be re- por ted here.

Methods and Materials

Enzyme source. Normal human skin from the upper abdominal region of corpses was prepared mechanically, cleaned from adjacent fat tissue and from loose scales, washed several times with cold saline, and cut into little pieces. Of these pieces, 1.0 g fresh weight was sus- pended in 5.0 ml iced saline and homogenized with a standard blendor (Ultraturrax) under cooling 3 times 30 sec with 1 rain intervals. After centrifugation (4°C, 5000 rpm for 15 rain), the supernatant was used for the incubation experiments. 1.8 ml of the supernatants were mixed with 0.2 ml of D-penicillamine in Ringer or 0.2 ml plain Ringer in the controls. 1 ml of the final solutions contained the enzymatic activity of 180 mg skin (wet weight). Altogether, homogenates of 10 different corpses and preparations were assayed.

D-Penieillamine. D-Penicillamine (Biochemie, Vienna, Austria) was dissolved in Ringer solution pH 7.4. 0.2 ml of the D-penicillamine solutions were mixed with 1.8 ml of the homo- genates; final concentrations of D-penieillamine amounted to 10 -4, 10 -3, 10 -2, 10 -1, 1, and ]0 mg/ml, corresponding to molar concentrations between 67 × 10 -5 and 67 mM/l.

Test assays and controls. 0.2 ml of D-peniciltamine solutions or 0.2 ml Ringer solutions in the controls were mixed with 1.8 ml of the homogenates snd incubated at 37°C for 1 hr. Enzymatic activities were compared in test assays and controls.

Determination o] enzymatic avtivities

I, actate dehydrogenase (EC 1.1.1.27; LDH): 0.05 ml of the samples were mixed with 2.5 ml of a 0.18 raM/1 NADH in 50 mMfl phosphate buffer pH 7.5 containing 0.6 mM/l pyruvate (temperature 25°C). Absorption at 340 nm was read immediately and after 1, 2, and 3 min. The average difference in extinction per rain was multiplied with 8200 to obtain mU LDH- activity per ml (y~Ioles × rain-Z).

Glucose.6-phosphate dehydrogenase (EC 1.1.1.49; G-6-PDH): 1.0 ml of the homogenate was mixed with 2.0 ml of a 0.05 M/1 triethanole amine buffer pH 7.6 containing 0.5 mM/1 ethylenediamine tetraacetate, 0.05 ml of a 31 raM/1 glucose-6-phosphate, and 0.1 ml of 10 mM/l NADP (temperature 25°C). Extinction at 340 nm was read immediately and after 1, 2, and 3 rain. The mean extinction difference per rain was multiplied with 506 to obtain mU G-6- PDH-activity per ml (y.Moles × rain-Z).

Glyceraldehade-3-phosphate dehydrogenase (EC 1.2.1.12; GAPDH): In a cuvette, the fol- lowing solutions were mixed at 25°C: 2.47 ml of a 0.1 M triethanolamine buffer pH 7.6, 0.I ml of a 32 raM1 ATP-solution, 0.2 mi of a 93 raM/1 glycerate-3-phosphate solution, 0.05 ml of a 12 raM/1 NADH solution, 0.1 ml of a 26 raM/1 ethylene diamine tetraacetate solution, 0.06 ml of a 0.1 M[l magnesium sulphate solution, and 0.01 ml of a 3-phosphoglycerate kinase sus- pension in 3.2 M]l ammonium sulphate solution (approx. 4000 U/ml). The reaction was started by adding 0.1 ml of the homogenates. Optical density at 340 nm was read after 1, 2, 3, and 4 rain.

D-Penicillamine in Dermatology 89

The mean extinction difference per min was multiplied with 48 in order to obtain the U GAPDH-activity per ml (mMoles × min-~).

Alkaline phosphatase (EC 3.1.3.1; AP): 0.05 ml of the homogenates were mixed at 25°C with 2.0 ml of a 50 raM/1 glycine buffer pH 10.5 containing 0.0 raM/1 magnesium chloride and 5.5. raM/1 sodium p-nitrophenylphosphate. 1 min after stirring, extinction at 405 nm was read; readings are repeated after 1, 2, and 3 rain. The mean extinction difference per rain was multi- plied with 2216 in order to obtain mU AP-activity per ml (~Moles × rain-l).

Acid phosphatase (EC 3.1.3.2; AcP): 0.2 ml of the homogenates were incubated with 1.0 ml of a 50 raM/1 citrate buffer pH 4.8 containing 5.5 mM sodium p-nitrophenylphosphate at 37°C for 30 min. The enzymatic reaction was then stopped by adding 10.0 ml of a 0.02 M/1 sodium hydroxide. Extinction difference at 405 nm was read against a blank in which the homogenate had been added after sodium hydroxide. By multiplying the increase in extinction with 101, mU AcP-aetivity per ml were obtained.

"Leucine aminopeptidase" (EC 3.4.1.2; LAP): 0.1 ml of the homogenates were incubated with 3.0 ml of a 0.05 M/1 phosphate buffer pH 7.2 containing 0.25 mg L-leueine-p-nitroanilide at 25°C for 30 min. Extinction difference at 405 nm be~ore and alter incubation was multiplied with 108 in order to obtain mU LAP-activity per rot.

Results Protein content o/the homogenates. The protein content of the homogenates as

determined by the Biuret method amounted to 0.3 g per 100 ml.

Enzymatic activities in the controls. I n all of the homogenates prepared, signi- ficant activities of the 6 enzymes determined in this s tudy were encountered. Mean activities in mU/ml homogenate amounted to the following values: L D H 1107, G-6-PDH 17, G A P D H 1543, AP 9, AcP 37, and L A P 6. (Values after 60 rain in- cubation.)

The influence o/D-peniciUamine. An incubation with D-penieillamine provoked significant changes in 4 of the 6 enzymatic activities investigated. LDH- and LAP- activities remained practically unchanged. Wi th G-6-PDH- and AcP-activities a dose-related act ivat ion was encountered; GAPDH- and AP-activities were in- hibited. As a typical example of the results obtained, the changes in the 6 dif- ferent enzymat ic activities of 1 homogenate (3-fold incubation, threefold deter- mination) are depicted in Fig. 1.

The hatched column indicates the concentration range of D-penicillamine obtainable in human skin in vivo.

Discussion

By various biochemical pathways, D-penicillamine may provoke changes in enzymat ic activities: chelation of impor tant cations, chelation of inhibiting cations, reaction with sulfhydril groups, reaction with aldehyde groups, depolymerization of macroglobulins, and incorporation into enzymatical ly active protein fractions. Such possibilities have already been shown in m a n y instances [8,15,25,29, 30, 31, 35,43,44,46]. More detailled investigations have been performed on the effect of / ) -penic i l lamin on G-6-PDH- [29], AP- [31], proline- and lysine-hydroxylase- [34], py ruva te kinase- [43] and collagenase- [7] activities.

The well-documented effects of D-penicillamine on collagen metabolism (changes in collagen synthesis, degradation, hydroxylat ion, crosslinking) [10,15, 25] are par t ly mediated by changes in enzymatic activities. I n this respect, the importance of Fe ++- and Mn++-ions for matura t ion of collagen must be stressed

90 W. Raab and B. Gmeiner

.5 loo

50

Contro[

50

-~ 100

\ %

e G-6-PDH

/ o ,.,,~ Acid P

LDH, LAP

\

\ .

~"e. . . . . . £ GAPDH A ~ ' ~ . . . - o A P

. . . .

I IHli I I I Log C ~ A 10 4 10 "z 10 4 10 0 101 rag/m{

Fig. 1. Changes in 6 different enzymatic activities of human skin homogenates in the presence of D-penicillamine (concentrations between 10 -4 and 10mg, ml, i.e. 67 × 10 -5 and 67 raM/l). Activation (upper part) and inactivation (lower part) is shown. All values in percent of controls

(incubation at 37 ° C for 60 miu)

[3, 36]. Furthermore, divalent cations have been shown to inhibit collagenolysis by lysosomal enzymes; D-penicillamine binds those cations and collagenolysis may develop uninhibited [1]. I t could be demonstrated on human dermal collagen that the normally occurring hydroxylation after exposure to ultraviolett light [26] is inhibited to a large degree when chelating agents are present [27].

Elastin formation, too, is changed by D-penicillamine as lysine-incorporation is disturbed and the copper-ions are removed [19].

The depression o] mesenchymaI reactions by D-penicillamine [22] is not only due to changes in collagen and elastin formation; direct inhibition of fibroblasts [14], of DNA- [18, 42] and of protein-synthesis [42] significantly contribute to this effect. Via similar mechanisms, the antifungal, antibacterial, and antiviral actions of D-peniciUamine may be explained [32,38,45].

In this study, changes in 4 of 6 enzymatic activities of human skin homogenates have been encountered following an incubation with D-penicillamine (Fig. 1 ). Such concentrations as they have been shown in this study to change enzymatic ac- tivities may be reached in vivo when patients are submitted to D-penicillamine treatment. Pharmacokinetic studies in rats revealed that 4 to 8°/0 of orally given D-penieiUamine is deposited in the skin [24,33]. 4°/o of 6.0 g amount to 240 mg which are distributed in 5 kg fresh weight of the human skin, making up con- centrations of about 50 ~g D-penicillamine per g fresh (wet) skin. A range of 50 to 100 ~g per g must be assumed therefore. For our assays which used 180 mg skin

D-Penicillamine in Dermatology 91

(wet weight) per ml, that would give a concentrations range of approximately 10 to 20 Ezg D-penicillamine per ml. At this range--as indicated by the hatched column in Fig. 1--significant changes in enzymatic activities were encountered.

By changes in enzymatic activities of the human skin, therapeutic as well as unwanted effects of D-penicillamin may be provoked.

In dermatological therapy, D-penicillamine which by far is better tolerated than other lathyrogenic agents e.g. ~-aminonitril [17] has been used in patients with scleroderma (progressive systemic sclerosis, extensive morphea); failures [9] and successes [13,21,40,41] have been reported. As therapeutic action of D- penicillamine in scleroderma the increase in solubility of collagen [10,11,15, 28, 47] as well as the depolymerization of the ground substance [6] were discussed. The administration of D-penicillamine to patients with disturbances of keratini- zation (e. g. Morbus Darier) should cause an ammelioration by the interference of the drug with cystine incorporation into keratin [2].

As unwanted dermatological effects of D-penicillamine, various lesions have been observed which altogether have been called the examples of human lathy- rism: pressure blisters, papules, inclusion cysts, and nail lesions [5,16, 39]. Further- more, fall in skin thickness [12], failure of wound healing [4], and elastosis per- forans serpiginosa [23] were reported in patients under prolonged treatment with D-penicillamine. A newborn child the mother of which had received D-penicill- amine in pregnancy exhibited congenital tissue defects such as lax skin, hyper- flexibility, vascular fragility, and impaired wound healing [20].

In addition to the direct effects of D-penicillamine such as chelation of cations, reactions with sulfhydril groups and aldehydes, indirect effects have to be con- sidered which are caused by changes in enzymatic activities. Such changes have been shown in this s tudy with concentrations of D-penicillamine obtainable under normal t reatment regimens in vivo.

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