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Healing of skin incision wounds treated with topically applied BAPN free base in the rat
EXPERIMENTAL
AND
Healing
MOLECULAR
PATHOLOGY
39, 154-162 (1983)
of Skin Incision Wounds Treated with Topically Applied BAPN Free Base in the Rat1
DARA L. HOFFMAN, JAMES A. OWEN AND MILOS Division
of Surgical
Biology,
Received
Department of Surgery, University Center, Tucson, Arizona 35724
October
30, 1981, and in revised
form
CHVAPIL~
of Arizona
April
Health
Sciences
11, 1983
P-Aminopropionitrile as free base (BAPN) was applied onto the incised or intact skin of rats at the dose of 5, 20, 100, and 200 pl for 9 days, twice daily. Breaking strength of the skin wound or intact skin was significantly reduced at doses of 20 pl and higher; body weight growth was significantly retarded at the two highest dosages. It is concluded that at a given dose (20 (~1) collagen polymerization (evaluated by reduced breaking strength and increased extractability of collagen) was specifically inhibited by BAPN. Furthermore, no evidence of topical or general toxic effects were observed, as reflected in histology, body weight growth, and behavior of the rats. Acute LDSu of BAPN base and fumarate, administered either ip or topically, was determined in mice. While BAPN base in ip administration shows LDsa of 1.15 g/kg, in cutaneous application it is more than 12.8 g/kg. It is suggested that topically applied BAPN base is percutaneously absorbed and affects collagen polymerization in the skin and adjacent tissues.
INTRODUCTION In the past 10 years our laboratory has been continuously involved in the search for effective and safe methods to interfere with pathological deposition or polymerization of collagen. Our leading concept stresses that it is not the amount of collagen which causes the abnormality of repair tissue, but rather the degree of collagen polymerization by stable covalent cross-links which results in scar contractures and strictures of fibrotic tissue (Chvapil, 1974a,b; Chvapil and Hurych, 1968). We have therefore focused our research on testing a drug which is probably the most effective inhibitor of collagen polymerization, B-aminopropionitrile fumarate (BAPN). BAPN is known to irreversibly inhibit the enzyme lysyl oxidase which oxidatively deaminates e-amino groups of lysyl and hydroxylysyl residues in the collagenous polypeptide chain. These formed aldehydes then become part of the covalent cross-link. Rather extensive research on BAPN fumarate clearly demonstrated its high effectiveness in various models of fibrotic lesions (for review see Chvapil, 1974a; Chvapil and Hurych, 1968 and Peacock, 1973) and the drug’s fast metabolism to cyanoacetic acid. As BAPN-depleted lysyl oxidase is rather quickly resynthesized, it is necessary to keep the concentration of this drug stable, which requires frequent administrations (Arem et al., 1979). Frequently, toxic side effects at lathyrogenic dosage levels of BAPN were observed. These effects are reflected in a slower growth rate and behavioral changes such as lethargy (Chvapil et al., 1979). To overcome drug toxicity, a combination of BAPN with another lathyrogen, D-penicillimine, was suggested (Haney et al., 1973). Each drug affects the polymerization of collagen by different mechanisms (Chvapil and Hurych, 1968). ’ Supported in part by Public Health Services Grant NS16133. ? To whom correspondence should be directed. 154 0014-4800183 $3.00 Copyright @ 1983 by Academic press, Inc. All rights of reproduction in any form reserved.
BAPN FREE BASE AND WOUND
HEALING
155
In another effort to reduce the toxic side effects of BAPN, we developed a drug delivery system for local administration (Speer et al., 1975). Unfortunately, to this date all carriers for BAPN tested by themselves showed tissue irritation. Recently we reported (Chvapil et al., 1980) that BAPN free base existing as a liquid readily penetrates the stratum corneum when administered topically and can be detected in the urine. It was also demonstrated that the topically applied drug inhibited lysyl oxidase in artificially induced subcutaneous granuloma tissue (Chvapil et al., 1980). We further reported the striking effect of topical administration of BAPN base on joint stiffness and peritendinous adhesions in rabbits and chickens, respectively (Karpman et al., 1981). We now report on the effectiveness and toxicity of topically applied BAPN free base, as tested in mouse and rat models. Dose-response data will be correlated with toxicological observations. METHODS Chemicals. B-Aminopropionitrile (BAPN) free base was purchased from Frinton Labs, New Jersey. The purity of the drug was analyzed by gas liquid chromatography. We found it to be 99.6% pure; 0.4% formed a not-yet-identified peak preceding the BAPN. B-Aminopropionitrile fumarate was purchased from Aldrich Chemical Company, Wisconsin. To consistently apply the same volume of BAPN free base, we diluted the original solution (13 54 M) with sterile water (Travinol USP, 2120302). The stability of diluted BAPN was determined using a Hewlett-Packard gas chromatograph, model 402, (column: 1.8 m; coating: 10% EGSS-X; column oven temperature: 190°C isothermal; injection port temperature: 255°C). The compound was found to have a retention time of 128 set and to be stable in HZ0 for at least 24 hr. Thus, fresh solutions were prepared daily. The effects of BAPN free base on wound healing were evaluated using 40 male 160-g Sprague-Dawley rats (Hilltop Breeding Labs). Animals were anesthetized with Innovar Vet (Pittman-Moore, 50002), shaved, and prepped. A 4.0-cm-long dorsal midline incision was made and approximated with four evenly spaced interrupted sutures (4-O Proline, Ethicon 8942). Topical applications of BAPN onto the wound area and caudally onto intact skin were started 24 hr postsurgitally and continued twice daily for 9 days. BAPN base (as a liquid) was evenly applied with a tuberculin syringe over 4-cm2 treatment areas. The same volume of saline was applied similarly to control animals. Details of the experiments are shown in the respective tables. The determinations of LDsO of BAPN free base and BAPN fumarate via ip and topical administrations were made using 80 male CD-l mice (Charles River Breeding Labs), with an average body weight of 20 g. Determination of LDs,, was performed as per the method of Weil (1952). Four dosage levels spaced in a geometric progression were evaluated using five animals at each dose. Animals receiving topical applications were shaved on the dorsal surfaced 24 hr prior to the treatment. After administration, the animals were observed for a 24-hr period for changes in behavior and number of mortalities. Breaking strength of the wound and intact skin was measured 10 days postsurgically. Treatment areas were excised in block; sutures, subdermal fascia, and panniculus were removed. Three O.Ccm-wide, full-thickness strips were cut per-
156
HOFFMAN,
OWEN, AND CHVAPIL
pendicular to the longitudinal axis of the wound. Three additional strips were cut from the intact skin. Breaking strength was determined using an Instron model TM Universal tensiometer at a rate of loading of 180 g/set. The extractability of collagen in the healing wounds was analyzed according to Jackson and Bentley (1960). Frozen skin tissue samples were pulverized using liquid nitrogen and extracted in 0.1 M acetic acid while shaking for 24 hr at 4°C. After acid hydrolysis of the soluble supernatent (6 N HCl, 10X, 18 hr), hydroxyproline was measured by the Stegemann (1958) procedure. Histology. Representative samples were evaluated by standard histological techniques using hematoxylin-eosin and Masson trichrome stains. Statistics. Duncan’s (1955) multiple range was used to test the significance of the data. RESULTS When 0.2 ml of BAPN free base was applied 24 hr after surgery and twice daily for a total of 9 days, the breaking strength of the wound (0.4-cm-wide strips) was significantly reduced from 219.5 to 72.1 g. The mean body weight of these animals on Day 9 was 170 g, while that of the control group was 220 g. The breaking strength of the wound was also significantly reduced when BAPN free base was applied for only the first or last 7 days of treatment. The body weights in these groups were also significantly lower than those of the controls (Table I). It was obvious that we were using an excessive dose of BAPN, as evidenced by a marked topical effect, formation of an excessive scab, and by an inhibition of body growth. We then tested the effect of 5, 20, and 100 p.1 of BAPN free base, applied in a total volume of 0.1 ml. The treatment was applied twice daily over the area of 4 cm2, including both the skin incision and extending caudally to the intact skin. The mean breaking strengths of the wounds were reduced to 174, 119, and 50 g, respectively, compared to the control mean of 206 g (Table II). This reduction was significant in those groups receiving 20 and 100 ~1 BAPN free base. A similar effect was obtained with the breaking strength of intact skin treated with 20 and 100 p.1 BAPN free base. Body weights were analyzed as a function of growth rate during the experiment (Fig. 1). This rate was significantly inhibited at the dosage levels of 100 ~JJBAPN free base (P < 0.05). Animals treated with 5 ~1 showed no inhibition of body growth rate when compared to controls. There was no statistical difference beTABLE I Effect of Topical BAPN Free Base on Breaking Strength of Skin Incision Wounds Group0 Control, saline 9 days Saline 2 days-BAPN 7 daysC BAPN 7 days=-saline 2 days BAPN 9 days’ u Treatment started 24 hr after surgery. b Initial body weight was 160 + 5 g. c BAPN 0.2 ml twice daily. dP < 0.05.
Body weigh@ (g) 220 197 180 170
c * 2 t
8.3 4.Sd 5.2d 5.7d
Breaking strength (g/O.4 cm) 219.5 77.2 89.9 72.1
2 e 2 ?
15.9 14Jd 1O.6d 18.6d
BAPN FREE BASE AND WOUND
HEALING
157
TABLE II Effect of BAPN Free Base on Skin Wound Characteristics
Dose of BAPN
Body weight 6s) Day 11
Liver wet weight (g)
Kidney wet weight (g)
Wound skin breaking % A.S.C.ldry strength (g/O.4 cm) weight
0 (Control) 5 20 100 200
247 2 3.5
9.5 2 0.01
2.7 f 0.17
206 f 20.1
11.2 2 0.2
2011 f 102
231 226 177 170
9.5 9.5 8.5 6.9
2.8 2.5 2.0 2.0
174 119 50 72
10.4 + 0.6 13.6 f 0.7* -
2198 1643 484 285
f f k -c
5.2 4.7* 4.3* 5.7*
+ 2 * 2
0.03 0.02 0.39* 0.2*
* + * k
0.05 0.07 0.05* 0.05*
” f f k
11.6 11.5* 6.7* 8.6*
Intact shin breaking strength (g/O.4 cm)
+ f f f
156 go* 88* 72*
Notes. Variability given as x 2 SEM, based on five animals per group. BAPN base applied in 0.1 ml volume. * P < 0.05.
tween the growth rates of animals treated with 5 or 20 ~1 of BAPN free base. In the 20-4 group, the rate was slightly affected, but falls within the normal variance of standard outbred SpragueDawley rats, compared at an identical period of the growth cycle (Hilltop Breeding Labs). Liver and kidney weights were not affected at 5 or 20 ~1, but were reduced significantly in the 100~~1 group (Table II). One overall effect of lathyrogenic inhibition of collagen cross-linking is an increase in the amount of soluble collagen. Thus, the comparative measurement of acid-extractable collagen is indicative of lathyrogenic collagen. Determinations were performed on excised wounds treated with saline (control) or with 5 and 20 ~1 of BAPN. Percentage of acid-soluble collagen was not affected in animals treated with 5 ~1 BAPN free base, but increased significantly in animals receiving
2.0 0.0-J
I
0.1 ml Saline FIG. 1. Effect of various topical doses of BAPN free base on body growth rate of rats. Values represent X f SD for growth rate as determined during the 11 days of the experiment. Shaded area represents percent of variance of body growth rate as determined in standard outbred S/D male rats.
158
BAPN FREE BASE AND WOUND
HEALING
159
20 ~1, when compared to controls (Table II). These data further support the biomechanical measurements of the wounded skin. Histological observations. The morphology of skin incision wounds as well as intact skin was compared in tissue samples treated with saline and with 20 and 100 p,l of BAPN free base (Figs. 2, 3). In each group at least five skin or skin wound specimens, each in three to five sections, were analyzed. There was no evidence of toxic topical effects with the 20-p,l dosage. The extent of epithelization of the wound, as well as the overall thickness of multilayered epithelial tissue, was the same as in controls; the epithelial cells were intact. There was no evidence of abnormal granulation tissue. In some sections of skin incision wounds treated with 20 ~1 BAPN free base, we observed slight edema in subepidermal space. No cellular inflammatory infiltrate was present. With 100 ~1 BAPN applied twice daily, both the wound and intact skin showed signs of irritation, toxicity, and scab formation. The epidermal layer was thinner and at certain areas completely necrotic and infiltrated with inflammatory cells. In the wound, granulation tissues were excessive and inflammatory infiltration was noticeable. Acute toxicity. The results of the acute toxicity study of BAPN fumarate and free base administered either ip or topically onto the skin are shown in Table III. BAPN fumarate has a low toxicity level of 3 g/kg ip. BAPN base is approximately 2.6 times more toxic than fumarate in ip administration. It is interesting that the acute LDSo of the metabolite of BAPN, cyanoacetic acid, is at least 10 times more toxic than the parent compound. We were not able to determine an actual LDSo for BAPN fumarate or free base on cutaneous administration. The volumes of the substance used were too large to allow meaningful testing. At the highest dosage of BAPN fumarate tested, 23.13 g/kg (corresponding to 12.8 g/kg of BAPN), no deaths or signs of toxicity were recorded within 24 hr after the administration. Only at the highest dosage of BAPN free base, 12.8 g/kg, 20% mortality occurred and transitory signs of toxicity, such as lethargy and dyspnea, were evident 2 to 19 hr after cutaneous application. DISCUSSION To our knowledge, BAPN as free base has never been tested in animals or used in human pathology as a drug. Only recently have we shown that BAPN base penetrates the stratum corneum barrier and is percutaneously absorbed (Chvapil et al., 1980). Thus, its application onto intact skin resulted in detection of the drug in urine. Based on our preliminary experience with the rate of percutaneous absorption of topically applied BAPN (Chvapil et al., 1980), we decided to apply the drug twice daily. Using this frequency of application and the dose of 0.1 ml per treatment, we showed significant improvement of peritendineous adhesions and joint stiffness in animal models (Karpman et al., 1981). In this study we showed that a much lower dose application (20 ~1) produced FIG. 2. Effect of various doses of BAPN free base administered on intact skin of rats. Top-control skin, treated with saline, middle-20 ~1 BAPN, bottom-100 ~1 BAPN. The skin was treated twice daily for 9 days. Approximately 40 x magnified. Note normal morphology at a 20-11.1dose. Thinning of epidermal layer, necrotic changes, inflammatory cell infiltrate, and eschar formation are present at a 100~~1 BAPN dose.
160
HOFFMAN,
OWEN,
AND
CHVAPIL
BAPN FREE BASE AND WOUND HEALING TABLE III BAPN Fumarate and BAPN Free Base LD-24
161
Hours
Substance
Wkd
LDjo cutaneous (g/k)
BAPN fumarate BAPN free base Cyanoacetic acide
2.968“ 1.152 0.200
>12.80bac >12.80“ -
LDso,
ip
0 LDsa refers to BAPN only; for fumarate it is 5.362 g/kg. b Refers to BAPN only; for fumarate it is 23.13 gikg. c For technical reasons, no large dose-volume could be tested. d Only 20% of the mice died within 24 hr. Signs of toxicity (lethargy, dyspnea) were present from 2 to 19 hr after cutaneous application. e According to “Registry of Toxic Effects of Chemical Substances, 1975.”
significant reduction of breaking strength of the incision wound as well as of the intact skin. This effect has been commonly ascribed to reduction in the formation of covalent intermolecular cross-links in collagenous structures, unless there is a generalized toxic effect, e.g., loss of body weight, which would also interfere with the synthesis and activity of lysyl oxidase (Madia et al., 1979). It is therefore important that at the dosage of 20 l~l(l9 mg BAPN) administered twice daily for 9 days, there was no effect on body growth; only the decreased collagen polymerization was significant. Thus, we conclude that the reduction of breaking strength is a specific topical effect of BAPN free base. There is no doubt that topically administered BAPN free base becomes toxic at certain concentrations-both locally and systemically. Topical signs manifest themselves as localized necrosis and excessive scab formation. We encountered the formation of scab over the incision wound and intact skin treated with 0.2 ml of BAPN, and to a lesser extent with the O.l-ml dosage. The two lower dosages did not induce any gross or microscopic changes in the wound tissue or intact skin. In view of the fact that the given dosages of BAPN free base were applied evenly over the wounded area and intact skin, we can assume that a smaller fraction of the dose would be sufficient to control the cross-linking strictly over the injured area. While the dosage of systemically administered BAPN fumarate (or hydrochloride) is commonly presented as milligrams per kilogram body weight, for topical skin applications the measure of the dosage should refer to the volume (dosage) spread over a certain area; thus, milligrams per square centimeter should best describe the dosage level. As the density of BAPN free base fluid is close to 1 (0.95), the volume corresponds closely to the weight of the administered drug. The results of this study indicate that the BAPN dosage of 10 mg/cm2 applied twice daily should be effective in reducing collagen polymerization without causing local or systemic toxic effects. It is obvious that the effect of BAPN base on FIG. 3. Effect of various dosages of BAPN free base on the morphology of skin wound. The picture refers to 11-day-old wound; top-saline treated, middle-20 ~1 BAPN, and bottom-100 ~1 BAPN treated. Magnification approximately 40 x . Note continuous epithelization over granulation tissue in control wound. After 20 p,l BAPN, well-developed and continuous epithelium covers the wound. Edema is present in the subepithelial space. At 100 (~1BAPN, excessive inflammatory cell infiltration is present. Epithelium lining is not closed. Over the wound a thick layer of eschar is found.
162
HOFFMAN,
OWEN,
AND
CHVAPIL
body weight growth is typical for our animal model (rat) with small absolute body mass. While topically applied BAPN free base at a high dosage level produced transitory signs of toxicity (lethargy, dyspnea), the equimolar high dose of fumarate was absent of any signs of toxicity. This indicates that BAPN fumarate was either less toxic or did not penetrate the skin. Other experiments (unpublished) have shown that the latter explanation is probably correct. Comparison of our data on the acute toxicities of ip administered BAPN fumarate and base with the previously reported LDso of cyanoacetic acid indicates that the systemic toxicity of BAPN may be related to its metabolite rather than to the parent compound itself. Studies of the pharmacokinetics of BAPN free base are presently under way. We still do not know how much of the drug is absorbed from the dose applied onto the skin or how quickly it is metabolized into CAA and excreted. The evidence that BAPN free base is percutaneously absorbed offers several possibilities for developing a sustained release of the drug for time-controlled delivery. REFERENCES AREM, A. J., MISIOROWSKI, R., and CHVAPIL, M. (1979). Effects of low-dose BAPN on wound healing. J. Surg.
Res. 27, 228-232.
CHVAPIL, M. (1974a). Pharmacology of fibrosis and tissue injury. Environ.
Health
Perspect.
9, 283-
294.
CHVAPIL, M. (1974b). Pharmacology of fibrosis: Definitions, limits, and perspectives. Life Sci. 16, 1345-1362. CHVAPIL, M., HAMEROFF, S. R., O’DEA, K., and PEACOCK, E. E. (1979). Local anesthetics and wound healing. .I. Surg. Res. 27, 367-371. CHVAPIL, M., and HURYCH, J. (1968). Control of collagen biosynthesis. In “International Review of Connective Tissue Research” (D. A. Hall, ed.), Vol. 4, pp. 67-196. CHVAPIL, M., PEACOCK, E. E., CARLSON, E. C., BLAU, S.. STEINBRONN, K., and MORTON, D. (1980). Colchicine and wound healing. J. Surg. Res. 28, 49-56. DUNCAN, D. B. (1955). Multiple range and multiple F test. Biometrics 11, l-42. HANEY, A. F., PEACOCK, E. E.. and MADDEN, J. W. (1973). The effect of multiple lathyrogenic agents upon wound healing in rats. Proc. Sot. Exp. Biol. Med. 142, 289-292. JACKSON, D. S., and BENTLEY, J. P. (1960). On the significance of the extractable collagens. .I. Biophys.
Biochem.
Cytol.
7, 37-42.
KARPMAN, R. R., SPEER, D. R., and CHVAPIL, M. (1981). Control of peritendinous adhesions and joint stiffness by a topical lathyrogen. Abstr. Trans. Orthop. Res. Sot. 6, 202. MADIA, A. M., ROZOVSKI, S. J., and KAGEN, H. M. (1979). Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation. Biochim. Biophys. Acta 585, 481-487. PEACOCK, E. E. (1973). Biologic frontiers in the control of healing. Amer. J. Surg. 126, 708-713. SPEER,D. P., PEACOCK, E. E., and CHVAPIL, M. (1975). The use of large molecular weight compounds to produce local lathyrism in healing wound. J. Surg. Res. 19, 169-173. STEGEMANN, H. (1958). Determination of hydroxyproline. Z. Physiol. Chem. 311, 41-45. WEIL, C. S. (1952). Tables for conveninent calculation of median-effective dose (LDSO or ED& and instructions in their use. Biometrics 8, 249-263.
AND
Healing
MOLECULAR
PATHOLOGY
39, 154-162 (1983)
of Skin Incision Wounds Treated with Topically Applied BAPN Free Base in the Rat1
DARA L. HOFFMAN, JAMES A. OWEN AND MILOS Division
of Surgical
Biology,
Received
Department of Surgery, University Center, Tucson, Arizona 35724
October
30, 1981, and in revised
form
CHVAPIL~
of Arizona
April
Health
Sciences
11, 1983
P-Aminopropionitrile as free base (BAPN) was applied onto the incised or intact skin of rats at the dose of 5, 20, 100, and 200 pl for 9 days, twice daily. Breaking strength of the skin wound or intact skin was significantly reduced at doses of 20 pl and higher; body weight growth was significantly retarded at the two highest dosages. It is concluded that at a given dose (20 (~1) collagen polymerization (evaluated by reduced breaking strength and increased extractability of collagen) was specifically inhibited by BAPN. Furthermore, no evidence of topical or general toxic effects were observed, as reflected in histology, body weight growth, and behavior of the rats. Acute LDSu of BAPN base and fumarate, administered either ip or topically, was determined in mice. While BAPN base in ip administration shows LDsa of 1.15 g/kg, in cutaneous application it is more than 12.8 g/kg. It is suggested that topically applied BAPN base is percutaneously absorbed and affects collagen polymerization in the skin and adjacent tissues.
INTRODUCTION In the past 10 years our laboratory has been continuously involved in the search for effective and safe methods to interfere with pathological deposition or polymerization of collagen. Our leading concept stresses that it is not the amount of collagen which causes the abnormality of repair tissue, but rather the degree of collagen polymerization by stable covalent cross-links which results in scar contractures and strictures of fibrotic tissue (Chvapil, 1974a,b; Chvapil and Hurych, 1968). We have therefore focused our research on testing a drug which is probably the most effective inhibitor of collagen polymerization, B-aminopropionitrile fumarate (BAPN). BAPN is known to irreversibly inhibit the enzyme lysyl oxidase which oxidatively deaminates e-amino groups of lysyl and hydroxylysyl residues in the collagenous polypeptide chain. These formed aldehydes then become part of the covalent cross-link. Rather extensive research on BAPN fumarate clearly demonstrated its high effectiveness in various models of fibrotic lesions (for review see Chvapil, 1974a; Chvapil and Hurych, 1968 and Peacock, 1973) and the drug’s fast metabolism to cyanoacetic acid. As BAPN-depleted lysyl oxidase is rather quickly resynthesized, it is necessary to keep the concentration of this drug stable, which requires frequent administrations (Arem et al., 1979). Frequently, toxic side effects at lathyrogenic dosage levels of BAPN were observed. These effects are reflected in a slower growth rate and behavioral changes such as lethargy (Chvapil et al., 1979). To overcome drug toxicity, a combination of BAPN with another lathyrogen, D-penicillimine, was suggested (Haney et al., 1973). Each drug affects the polymerization of collagen by different mechanisms (Chvapil and Hurych, 1968). ’ Supported in part by Public Health Services Grant NS16133. ? To whom correspondence should be directed. 154 0014-4800183 $3.00 Copyright @ 1983 by Academic press, Inc. All rights of reproduction in any form reserved.
BAPN FREE BASE AND WOUND
HEALING
155
In another effort to reduce the toxic side effects of BAPN, we developed a drug delivery system for local administration (Speer et al., 1975). Unfortunately, to this date all carriers for BAPN tested by themselves showed tissue irritation. Recently we reported (Chvapil et al., 1980) that BAPN free base existing as a liquid readily penetrates the stratum corneum when administered topically and can be detected in the urine. It was also demonstrated that the topically applied drug inhibited lysyl oxidase in artificially induced subcutaneous granuloma tissue (Chvapil et al., 1980). We further reported the striking effect of topical administration of BAPN base on joint stiffness and peritendinous adhesions in rabbits and chickens, respectively (Karpman et al., 1981). We now report on the effectiveness and toxicity of topically applied BAPN free base, as tested in mouse and rat models. Dose-response data will be correlated with toxicological observations. METHODS Chemicals. B-Aminopropionitrile (BAPN) free base was purchased from Frinton Labs, New Jersey. The purity of the drug was analyzed by gas liquid chromatography. We found it to be 99.6% pure; 0.4% formed a not-yet-identified peak preceding the BAPN. B-Aminopropionitrile fumarate was purchased from Aldrich Chemical Company, Wisconsin. To consistently apply the same volume of BAPN free base, we diluted the original solution (13 54 M) with sterile water (Travinol USP, 2120302). The stability of diluted BAPN was determined using a Hewlett-Packard gas chromatograph, model 402, (column: 1.8 m; coating: 10% EGSS-X; column oven temperature: 190°C isothermal; injection port temperature: 255°C). The compound was found to have a retention time of 128 set and to be stable in HZ0 for at least 24 hr. Thus, fresh solutions were prepared daily. The effects of BAPN free base on wound healing were evaluated using 40 male 160-g Sprague-Dawley rats (Hilltop Breeding Labs). Animals were anesthetized with Innovar Vet (Pittman-Moore, 50002), shaved, and prepped. A 4.0-cm-long dorsal midline incision was made and approximated with four evenly spaced interrupted sutures (4-O Proline, Ethicon 8942). Topical applications of BAPN onto the wound area and caudally onto intact skin were started 24 hr postsurgitally and continued twice daily for 9 days. BAPN base (as a liquid) was evenly applied with a tuberculin syringe over 4-cm2 treatment areas. The same volume of saline was applied similarly to control animals. Details of the experiments are shown in the respective tables. The determinations of LDsO of BAPN free base and BAPN fumarate via ip and topical administrations were made using 80 male CD-l mice (Charles River Breeding Labs), with an average body weight of 20 g. Determination of LDs,, was performed as per the method of Weil (1952). Four dosage levels spaced in a geometric progression were evaluated using five animals at each dose. Animals receiving topical applications were shaved on the dorsal surfaced 24 hr prior to the treatment. After administration, the animals were observed for a 24-hr period for changes in behavior and number of mortalities. Breaking strength of the wound and intact skin was measured 10 days postsurgically. Treatment areas were excised in block; sutures, subdermal fascia, and panniculus were removed. Three O.Ccm-wide, full-thickness strips were cut per-
156
HOFFMAN,
OWEN, AND CHVAPIL
pendicular to the longitudinal axis of the wound. Three additional strips were cut from the intact skin. Breaking strength was determined using an Instron model TM Universal tensiometer at a rate of loading of 180 g/set. The extractability of collagen in the healing wounds was analyzed according to Jackson and Bentley (1960). Frozen skin tissue samples were pulverized using liquid nitrogen and extracted in 0.1 M acetic acid while shaking for 24 hr at 4°C. After acid hydrolysis of the soluble supernatent (6 N HCl, 10X, 18 hr), hydroxyproline was measured by the Stegemann (1958) procedure. Histology. Representative samples were evaluated by standard histological techniques using hematoxylin-eosin and Masson trichrome stains. Statistics. Duncan’s (1955) multiple range was used to test the significance of the data. RESULTS When 0.2 ml of BAPN free base was applied 24 hr after surgery and twice daily for a total of 9 days, the breaking strength of the wound (0.4-cm-wide strips) was significantly reduced from 219.5 to 72.1 g. The mean body weight of these animals on Day 9 was 170 g, while that of the control group was 220 g. The breaking strength of the wound was also significantly reduced when BAPN free base was applied for only the first or last 7 days of treatment. The body weights in these groups were also significantly lower than those of the controls (Table I). It was obvious that we were using an excessive dose of BAPN, as evidenced by a marked topical effect, formation of an excessive scab, and by an inhibition of body growth. We then tested the effect of 5, 20, and 100 p.1 of BAPN free base, applied in a total volume of 0.1 ml. The treatment was applied twice daily over the area of 4 cm2, including both the skin incision and extending caudally to the intact skin. The mean breaking strengths of the wounds were reduced to 174, 119, and 50 g, respectively, compared to the control mean of 206 g (Table II). This reduction was significant in those groups receiving 20 and 100 ~1 BAPN free base. A similar effect was obtained with the breaking strength of intact skin treated with 20 and 100 p.1 BAPN free base. Body weights were analyzed as a function of growth rate during the experiment (Fig. 1). This rate was significantly inhibited at the dosage levels of 100 ~JJBAPN free base (P < 0.05). Animals treated with 5 ~1 showed no inhibition of body growth rate when compared to controls. There was no statistical difference beTABLE I Effect of Topical BAPN Free Base on Breaking Strength of Skin Incision Wounds Group0 Control, saline 9 days Saline 2 days-BAPN 7 daysC BAPN 7 days=-saline 2 days BAPN 9 days’ u Treatment started 24 hr after surgery. b Initial body weight was 160 + 5 g. c BAPN 0.2 ml twice daily. dP < 0.05.
Body weigh@ (g) 220 197 180 170
c * 2 t
8.3 4.Sd 5.2d 5.7d
Breaking strength (g/O.4 cm) 219.5 77.2 89.9 72.1
2 e 2 ?
15.9 14Jd 1O.6d 18.6d
BAPN FREE BASE AND WOUND
HEALING
157
TABLE II Effect of BAPN Free Base on Skin Wound Characteristics
Dose of BAPN
Body weight 6s) Day 11
Liver wet weight (g)
Kidney wet weight (g)
Wound skin breaking % A.S.C.ldry strength (g/O.4 cm) weight
0 (Control) 5 20 100 200
247 2 3.5
9.5 2 0.01
2.7 f 0.17
206 f 20.1
11.2 2 0.2
2011 f 102
231 226 177 170
9.5 9.5 8.5 6.9
2.8 2.5 2.0 2.0
174 119 50 72
10.4 + 0.6 13.6 f 0.7* -
2198 1643 484 285
f f k -c
5.2 4.7* 4.3* 5.7*
+ 2 * 2
0.03 0.02 0.39* 0.2*
* + * k
0.05 0.07 0.05* 0.05*
” f f k
11.6 11.5* 6.7* 8.6*
Intact shin breaking strength (g/O.4 cm)
+ f f f
156 go* 88* 72*
Notes. Variability given as x 2 SEM, based on five animals per group. BAPN base applied in 0.1 ml volume. * P < 0.05.
tween the growth rates of animals treated with 5 or 20 ~1 of BAPN free base. In the 20-4 group, the rate was slightly affected, but falls within the normal variance of standard outbred SpragueDawley rats, compared at an identical period of the growth cycle (Hilltop Breeding Labs). Liver and kidney weights were not affected at 5 or 20 ~1, but were reduced significantly in the 100~~1 group (Table II). One overall effect of lathyrogenic inhibition of collagen cross-linking is an increase in the amount of soluble collagen. Thus, the comparative measurement of acid-extractable collagen is indicative of lathyrogenic collagen. Determinations were performed on excised wounds treated with saline (control) or with 5 and 20 ~1 of BAPN. Percentage of acid-soluble collagen was not affected in animals treated with 5 ~1 BAPN free base, but increased significantly in animals receiving
2.0 0.0-J
I
0.1 ml Saline FIG. 1. Effect of various topical doses of BAPN free base on body growth rate of rats. Values represent X f SD for growth rate as determined during the 11 days of the experiment. Shaded area represents percent of variance of body growth rate as determined in standard outbred S/D male rats.
158
BAPN FREE BASE AND WOUND
HEALING
159
20 ~1, when compared to controls (Table II). These data further support the biomechanical measurements of the wounded skin. Histological observations. The morphology of skin incision wounds as well as intact skin was compared in tissue samples treated with saline and with 20 and 100 p,l of BAPN free base (Figs. 2, 3). In each group at least five skin or skin wound specimens, each in three to five sections, were analyzed. There was no evidence of toxic topical effects with the 20-p,l dosage. The extent of epithelization of the wound, as well as the overall thickness of multilayered epithelial tissue, was the same as in controls; the epithelial cells were intact. There was no evidence of abnormal granulation tissue. In some sections of skin incision wounds treated with 20 ~1 BAPN free base, we observed slight edema in subepidermal space. No cellular inflammatory infiltrate was present. With 100 ~1 BAPN applied twice daily, both the wound and intact skin showed signs of irritation, toxicity, and scab formation. The epidermal layer was thinner and at certain areas completely necrotic and infiltrated with inflammatory cells. In the wound, granulation tissues were excessive and inflammatory infiltration was noticeable. Acute toxicity. The results of the acute toxicity study of BAPN fumarate and free base administered either ip or topically onto the skin are shown in Table III. BAPN fumarate has a low toxicity level of 3 g/kg ip. BAPN base is approximately 2.6 times more toxic than fumarate in ip administration. It is interesting that the acute LDSo of the metabolite of BAPN, cyanoacetic acid, is at least 10 times more toxic than the parent compound. We were not able to determine an actual LDSo for BAPN fumarate or free base on cutaneous administration. The volumes of the substance used were too large to allow meaningful testing. At the highest dosage of BAPN fumarate tested, 23.13 g/kg (corresponding to 12.8 g/kg of BAPN), no deaths or signs of toxicity were recorded within 24 hr after the administration. Only at the highest dosage of BAPN free base, 12.8 g/kg, 20% mortality occurred and transitory signs of toxicity, such as lethargy and dyspnea, were evident 2 to 19 hr after cutaneous application. DISCUSSION To our knowledge, BAPN as free base has never been tested in animals or used in human pathology as a drug. Only recently have we shown that BAPN base penetrates the stratum corneum barrier and is percutaneously absorbed (Chvapil et al., 1980). Thus, its application onto intact skin resulted in detection of the drug in urine. Based on our preliminary experience with the rate of percutaneous absorption of topically applied BAPN (Chvapil et al., 1980), we decided to apply the drug twice daily. Using this frequency of application and the dose of 0.1 ml per treatment, we showed significant improvement of peritendineous adhesions and joint stiffness in animal models (Karpman et al., 1981). In this study we showed that a much lower dose application (20 ~1) produced FIG. 2. Effect of various doses of BAPN free base administered on intact skin of rats. Top-control skin, treated with saline, middle-20 ~1 BAPN, bottom-100 ~1 BAPN. The skin was treated twice daily for 9 days. Approximately 40 x magnified. Note normal morphology at a 20-11.1dose. Thinning of epidermal layer, necrotic changes, inflammatory cell infiltrate, and eschar formation are present at a 100~~1 BAPN dose.
160
HOFFMAN,
OWEN,
AND
CHVAPIL
BAPN FREE BASE AND WOUND HEALING TABLE III BAPN Fumarate and BAPN Free Base LD-24
161
Hours
Substance
Wkd
LDjo cutaneous (g/k)
BAPN fumarate BAPN free base Cyanoacetic acide
2.968“ 1.152 0.200
>12.80bac >12.80“ -
LDso,
ip
0 LDsa refers to BAPN only; for fumarate it is 5.362 g/kg. b Refers to BAPN only; for fumarate it is 23.13 gikg. c For technical reasons, no large dose-volume could be tested. d Only 20% of the mice died within 24 hr. Signs of toxicity (lethargy, dyspnea) were present from 2 to 19 hr after cutaneous application. e According to “Registry of Toxic Effects of Chemical Substances, 1975.”
significant reduction of breaking strength of the incision wound as well as of the intact skin. This effect has been commonly ascribed to reduction in the formation of covalent intermolecular cross-links in collagenous structures, unless there is a generalized toxic effect, e.g., loss of body weight, which would also interfere with the synthesis and activity of lysyl oxidase (Madia et al., 1979). It is therefore important that at the dosage of 20 l~l(l9 mg BAPN) administered twice daily for 9 days, there was no effect on body growth; only the decreased collagen polymerization was significant. Thus, we conclude that the reduction of breaking strength is a specific topical effect of BAPN free base. There is no doubt that topically administered BAPN free base becomes toxic at certain concentrations-both locally and systemically. Topical signs manifest themselves as localized necrosis and excessive scab formation. We encountered the formation of scab over the incision wound and intact skin treated with 0.2 ml of BAPN, and to a lesser extent with the O.l-ml dosage. The two lower dosages did not induce any gross or microscopic changes in the wound tissue or intact skin. In view of the fact that the given dosages of BAPN free base were applied evenly over the wounded area and intact skin, we can assume that a smaller fraction of the dose would be sufficient to control the cross-linking strictly over the injured area. While the dosage of systemically administered BAPN fumarate (or hydrochloride) is commonly presented as milligrams per kilogram body weight, for topical skin applications the measure of the dosage should refer to the volume (dosage) spread over a certain area; thus, milligrams per square centimeter should best describe the dosage level. As the density of BAPN free base fluid is close to 1 (0.95), the volume corresponds closely to the weight of the administered drug. The results of this study indicate that the BAPN dosage of 10 mg/cm2 applied twice daily should be effective in reducing collagen polymerization without causing local or systemic toxic effects. It is obvious that the effect of BAPN base on FIG. 3. Effect of various dosages of BAPN free base on the morphology of skin wound. The picture refers to 11-day-old wound; top-saline treated, middle-20 ~1 BAPN, and bottom-100 ~1 BAPN treated. Magnification approximately 40 x . Note continuous epithelization over granulation tissue in control wound. After 20 p,l BAPN, well-developed and continuous epithelium covers the wound. Edema is present in the subepithelial space. At 100 (~1BAPN, excessive inflammatory cell infiltration is present. Epithelium lining is not closed. Over the wound a thick layer of eschar is found.
162
HOFFMAN,
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AND
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body weight growth is typical for our animal model (rat) with small absolute body mass. While topically applied BAPN free base at a high dosage level produced transitory signs of toxicity (lethargy, dyspnea), the equimolar high dose of fumarate was absent of any signs of toxicity. This indicates that BAPN fumarate was either less toxic or did not penetrate the skin. Other experiments (unpublished) have shown that the latter explanation is probably correct. Comparison of our data on the acute toxicities of ip administered BAPN fumarate and base with the previously reported LDso of cyanoacetic acid indicates that the systemic toxicity of BAPN may be related to its metabolite rather than to the parent compound itself. Studies of the pharmacokinetics of BAPN free base are presently under way. We still do not know how much of the drug is absorbed from the dose applied onto the skin or how quickly it is metabolized into CAA and excreted. The evidence that BAPN free base is percutaneously absorbed offers several possibilities for developing a sustained release of the drug for time-controlled delivery. REFERENCES AREM, A. J., MISIOROWSKI, R., and CHVAPIL, M. (1979). Effects of low-dose BAPN on wound healing. J. Surg.
Res. 27, 228-232.
CHVAPIL, M. (1974a). Pharmacology of fibrosis and tissue injury. Environ.
Health
Perspect.
9, 283-
294.
CHVAPIL, M. (1974b). Pharmacology of fibrosis: Definitions, limits, and perspectives. Life Sci. 16, 1345-1362. CHVAPIL, M., HAMEROFF, S. R., O’DEA, K., and PEACOCK, E. E. (1979). Local anesthetics and wound healing. .I. Surg. Res. 27, 367-371. CHVAPIL, M., and HURYCH, J. (1968). Control of collagen biosynthesis. In “International Review of Connective Tissue Research” (D. A. Hall, ed.), Vol. 4, pp. 67-196. CHVAPIL, M., PEACOCK, E. E., CARLSON, E. C., BLAU, S.. STEINBRONN, K., and MORTON, D. (1980). Colchicine and wound healing. J. Surg. Res. 28, 49-56. DUNCAN, D. B. (1955). Multiple range and multiple F test. Biometrics 11, l-42. HANEY, A. F., PEACOCK, E. E.. and MADDEN, J. W. (1973). The effect of multiple lathyrogenic agents upon wound healing in rats. Proc. Sot. Exp. Biol. Med. 142, 289-292. JACKSON, D. S., and BENTLEY, J. P. (1960). On the significance of the extractable collagens. .I. Biophys.
Biochem.
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7, 37-42.
KARPMAN, R. R., SPEER, D. R., and CHVAPIL, M. (1981). Control of peritendinous adhesions and joint stiffness by a topical lathyrogen. Abstr. Trans. Orthop. Res. Sot. 6, 202. MADIA, A. M., ROZOVSKI, S. J., and KAGEN, H. M. (1979). Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation. Biochim. Biophys. Acta 585, 481-487. PEACOCK, E. E. (1973). Biologic frontiers in the control of healing. Amer. J. Surg. 126, 708-713. SPEER,D. P., PEACOCK, E. E., and CHVAPIL, M. (1975). The use of large molecular weight compounds to produce local lathyrism in healing wound. J. Surg. Res. 19, 169-173. STEGEMANN, H. (1958). Determination of hydroxyproline. Z. Physiol. Chem. 311, 41-45. WEIL, C. S. (1952). Tables for conveninent calculation of median-effective dose (LDSO or ED& and instructions in their use. Biometrics 8, 249-263.