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Safety of lysosubtilin per os in mice, rabbits and calvesGa Biziulevichius, Ig Arestov

To cite this version:Ga Biziulevichius, Ig Arestov. Safety of lysosubtilin per os in mice, rabbits and calves. VeterinaryResearch, BioMed Central, 1997, 28 (4), pp.385-395. <hal-00902488>

Original article

Safety of lysosubtilin per os in mice, rabbits and calves

GA Biziulevichius IG Arestov

I Institute of’IllIllIunologB’, Sector ollllllllunohiotechnology, 12 Mok,vliiii;iki( St, 2600 Vilnius, Lithuania:2 Vitehsk Sttil(, Acwlel7lB’ < >f’ VW eri»arv Medicine. f)epal’tl7lellt of l»tornal A’«/;//!/ff7;!;<.s /.)).s’<!<B!.s

and PI/(/rl7lacologB&dquo; 7/// 1st f)ovatora St, Vitehsk, 2106/9 BB’elarus

(Received 21 October 1996; accepted 25 February 1997)

Summary - Lysosubtilin is a broad-spectrum preparation ol’lytic enzymes from Bacillus suhtilisdesigned for veterinary medicine as an alternative to common antibiotics. The safety of lysosubtilinwas studied in acute and subchronic toxicity tests in mice, rabbits, and calves. No sign of toxicosiswas observed when lyaosubtilin was administered as a single dose per os to white mice in dosesreaching 2 x 10!’ U/kg of weight. Subcutaneous LDStr ot’lysosubtilin in mice was equal to 5 x 105 U/kgof weight. In order to determine subchronic toxicity in mice and rabbits the doses of lysosubtilinused in veterinary practice (2 x I ()4 U/kg of weight) were increased up to ten times (2, 8, 20. 10’ U/kgof weight) and when experimenting with calves they were increased up to two times (4.1 ()4 U/kg ofweight) while extending the administration period by three times. In the three animal species, givenin increased doses twice a day per os for 30 days, lysosubtilin did not cause any evident signs ofclinical toxicosis. The blood and sera indices of rabbits and calves were within the normal range. Incalves the changes were insignificant. The ANOVA test, however, revealed statistically significantdifferences in hemoglobin, WBC, protein, and glucose value changes between rabbit groups whichwere given different doses of Iysosubtilin. A significant increase in daily weight gain (approximately100 g. P < 0.001 ), influenccd by lysosubtilin, was observed in calves. No deviations from the normalstatus were observed in the organs and tissues obtained during the post-mortem examination of rab-bits atnd calves slaughtered I and 15 days after the end of Iysosubtilin administration. Veterinary-san-itary examination ol the slaughtered animals and laboratory tests of their meat showed normal com-mcrcial and sanitary qualities.

lysosubtilin / safety / mouse / rabbit / newborn calf

Résumé &horbar; Innocuité de la lysosubtiline administrée par voie orale à des souris, des lapins et desveaux. La lysosubtiline est une préparation à large spectre d’enzymes lytiques de Bncillus .;fil>iili.;conçue pour la médecine vétérinaire, comme méthode alternative à l’utilisation d’antibiotiques com-muns. Chez la souris, le lapin et le veau, l’innocuité de la lysosubtiline a été étudiée lors des expériencesde toxicité aiguë et subchronique. Une dose de 2 x 106 U/kg de lysosubtiline per os en une seule foisà des souris blanches n’a entraîné aucun effet toxique. Injectée sous la peau, la dose létale 50 était

k Correspondence and reprintsTel: (370) 2 589224; fax: (370) 2 729827.

5 x 105 U/kg. Dans le but de déterminer la toxicité subchronique vis-à-vis de souris et de lapins, lesdoses de lysosubtiline utilisées en pratique vétérinaire (2 x 10-1 U/kg de poids) ont été augmentéesjusqu’à dix fois (2, 8 et 20 x 10! U/kg de poids) et deux fois dans les expériences sur les veaux(4 x 10-1 U/kg de poids), en prolongeant le temps d’injection de trois fois. La lysoaubtiline adminis-trée deux fois par jour per os pendant une période de 30 jours à des doses augmentées n’a provoquéaucun effet clinique de toxicité. Chez les lapins et les veaux, les paramètres sanguins et sériquessont restés dans les limites de la norme. Chez les veaux, leurs changements n’étaient pas statisti-quement significatifs. Le test ANOVA a révélé des différences statistiquement significatives entre tesgroupes de lapins pour les valeurs de F hémoglobine, des globules blancs, du glucose, selon les dosesde lysostibtiline. Une augmentation significative du gain de poids quotidien (sous l’effet de la lyso-aubtiline) a été observée chez les veaux (approximativement 100 g, p < 0.001 ). Lors d’examens

post-iiioileiii de lapins et de veaux, abattus 1 et 15 jours après l’administration, aucune modificationdes organes et des tissus n’a été démontréc. L’examen vétérinaire et sanitaire des animaux abattus etles tests en laboratoire ont confirmé une haute yualité sanitaire de la viande commercialisée.

lysosubtiline / innocuité / souris / lapin / veau nouveau-né

INTRODUCTION

Lytic enzymes or enzymes that degrade thecell walls of microorganisms arc a group ofantimicrobial agents differing from antibi-otics by their mode of action (Ghuysen etal, 1966; Strominger and Ghuysen, 1967;Phafll 1977; Zakharova and Pavlova, 1985). ).These enzymes (proteins in chemical nature)may successfully be applied for lysis ofantibiotic-resistant cultures (Severin andTomasz, 1995). Some lytic enzyme prepa-rations (eg, lysozynies of animal and bac-terizil origin, lysoainid!ise, lysostaphiii,mutanolysin) have been shown to be highlyeffective in human and/or animal medicine

(Zygmunt and Tavormina, 1972; Proctorand Cunningham, 1988; Blackbum and Pol-lack, 1990; Kislukhina et al, 1990; Skryabinand Kulaev, 1990).

Lysosubtilin is a partly purified broadantimicrobial spectrum enzyme preparationdesigned especially for veterinary medicineas an alternative to common antibiotics sinceconcerns about the undesirable after-effectsof the latter are increasing every year (Ara-neo et al, 1996; Piddock, 1996). It is one ofthe most promising preparations of lyticenzymes ever used in veterinary practice(Biziulevichius and Kislukhina, 1988). Itsphysicochemical properties, complex struc-ture as well as in vitro action on bacteria

and fungi have already been described (Biz-iulevichius et al, 1987, 1989, 1995).

Veterinary lysosubtiliii has been pro-duced on an industrial scale solely in Lithua-nia since 1983 (another type of lysosubtilin nfor the needs of microbiological industry iseven older). Since 1990, it has been listed

among the preparations approved for use inveterinary medicine throughout the formerSoviet Union (FSU). In independent Estonia.Latvia and Lithuania, its application wasreapproved in 1993.

The prophylaxis techniques and treat-ment of gastrointestinal disorders in new-born calves with lysosubtilin given per osas an antimicrobial agent as well as its use asa device to compensate for digestive enzymedeficiency and the resultant high efficacyhave already been described (Biziulevichiusand Arestov, 1997). In spite of the above-mentioned facts and the widespread use ofthe preparation in FSU, including the BalticStates (our calculations show that approxi-mately, 700 000 newborn calves were pro-tected or treated with lysosubtilin), there arestill sceptics who have doubts about thesafety of lysosubtilin. These concerns ham-per the spread of Iysosubtilin to other coun-tries, where the legislation safeguarding ani-mal health is stricter (Batza, 1995). It mustbe admitted that the results of lysosubliliii

safety studies when used per os for prophy-laxis and treatment of gastrointestinal dis-orders in newborn calves have never beendescribed, nor has the safety of similarenzyme preparations of the same destina-tion bacterial lysozyme and pepsinorm beenshown (Abramov and Shevchenko, 1991 ).

This paper provides the results of IYSO-subtilin safcty studies.

MATERIALS AND METHODS

The animal experiments were allowed and sup-ported by Veterinary Medicine Boards of Bye-larus and Lithuania. Safety evaluation of lyso-subtilin was performed according to the regulatoryguidelines for the experimental study of newenzytne preparations intended for clinical trials(anonymous, 1985) approved by Pharmacologi-cttl Committee of FSU Ministry of Health Care.

Animals and experimental procedures

Rationale behind the experiments

The prophylactic dose of Iysosubtilin for calf gas-trointestinal disorders administered twice a day for10 days is 2 x 10-1 U/kg of weight. The usutdduration of therapy applying a similar dose oflysosubtilin twice daily is 2-3 days (Biziulevichiusand Arestov, 1997). Thereforc in order to deter-minc subchronic toxicity in laboratory animals(mice and rabbits). the doses were increased up toten times and in experimenting with calves up totwo times while extending the longest period ofadministration of the preparation by three times.

Mouse experiments

Experiments were performed on CC57W micein the Department of Pharmacology of the Med-ical Faculty of Vilnius University (Lithuania).The mice, of I S-22 g body wcight, were kept ina room conditioned at 20 ± I °C and 55 ± 5<% in nrelative humidity and received standardized food.Both the animals and food were obtained fromthe Institute of Immunology (Vilnius, Lithua-nia). Drinking water was givcn ad libitumthroughout the experiments. In acutc oral andsubcutaneous toxicity studies, Iysosubtilin was

administered after being dissolved in distilledwater, while in subchronic oral toxicity studies itwas mixed into the powdered food.

For determination of the acute oral toxicityfour groups of mice were used (three for studyand one for control). Each group contained tenanimals (five males and 1-ive females). Lysosub-tilin was administered to the study group ani-mals as a single dose, the doses being I , 1.5 and2 x 106 U/kg of weight. All animals were sub-jected to daily clinical observation for 14 days.

For determination of the acute subcutaneous

toxicity, six groups of mice were used (fivc forstudy and one for control ). Each group contained12 animals (six males and six females). Lysosub-tilin was administered to the study group animalsby a aingle subcutaneous injection, the doses being0.5, 2, 7.5, I and 50 x 105 U/kg of weight. Thecontrol animals were injectcd with distilled wateras placebo. All animals were subjected to dailyclinical observation. The LD50 was calculated,

according to Litchfield and Wilcoxon ( 1949). fromthe mortality rate of mice on day 14.

For determination of the subchronic toxicityfour groups of mice were used (three for studyand one for control). Each group contained 20males. Lysosubtilin was given to the study groupanimals with food twice a day, the doses being 2,8 and 20 x 10‘t U/kg of weight, for 30 days. Allanimals were subjected to daily clinical obser-vation for 60 days. Body weight was measured onday 0, IS and 30. Determination of blood andscra indices, as not obligntive in mouse experi-ments according to the regulatory guidelines(anonymous, 1985), was not performed.

Rabbit experiments

Experiments were performed on Chinchilla rab-bits in the Department of Intcrnal NoninfectiousDiseases and Pharmacology of Vitebsk StateAcademy of Veterinary Medicine (Byelarus) andits training farrn. Twenty-four clinically healthymales. approximately 3 kg of weight ench, wereused. The animals were divided into four groups(three for study and one for control) ot- six ani-mals. Lysosubtilin was fed to each of the ani-mals with food twice a day, the doses being 2, 8and 20 x 104 U/kg of weight, for 30 days. Therabbits of the control group were not l’ed anylysosubtiliii. All animals were subjected to dailyclinical observation. The animals were also exam-ined more intensively at the beginning of theexperiment and on every tenth day. This exami-

nation included weighing and the determinationof blood and sera indices.

Calf experiments

The experiments were performed on LithuanianBrown breed newborn calves at the experimentalfarm located in the Zarasai region of Lithuania.Thirty calves were used simultaneously. Thecalves were kept in separate sheds and tendedcarefully. One group (15 calves) was fed withlysosubtilin (the dose being 4 x 104 U/kg ofweight) in colostrum or later milk, both supple-mented with 10 g/L of sodium bicarbonate begin-ning with the first feeding (30-45 min after birth)and then later twice a day for 30 days. The othergroup of IS calves (control) was fed accordingto the scheme usually applied in Lithuania andByelarus for prevention of diarrhea ( Valionis andMusteikis, 1980). Briefly the newborn calveswere fed with colostrum or later milk, electrolytes(Errovitini or So/ Ringeri-Locke), linseed mucilage(Mt«. ;7a!;.’..s’f/;;f;7;B Lini) and probiotics (usuallybroth containing lactobacilli) interchangeably,for 30 days. All animals were subjected to dailyclinical observation. At the beginning of theexperiment we randomly selected six calves fromeach of the two groups and measured their dailyweight gain over the 30 days. Blood and seraindices were determined in these calves on day 10,20 and 30 of the experiment.

On the third day of the experiment, signs ofdiarrhea were noticed in three calves from thecontrol group in spite of the protective measurestaken and these animals were excluded from fur-ther studies.

Lysosubtilin

Lysosubtilin (more precisely Lysosubtilin G10X),a commercial preparation of lytic enzymes fromBucillus subtilis SK-52, was acquired from thestate joint-stock enterprise Biosinteze (Vilnius,Lithuania). Lysosubtilin is obtained by cultivat-ing the producer in nutrient medium. Then theculture liquid is processed with calcium phos-phate gel and filtered. Filtrate is concentrated,the enzyme is precipitated with acetone and thefinal product is dried. It is a powder of light (grey-ish or yellowish) colour with a lytic activity of1 000 000 U/o according to Kisiukhina ( 1976).Unit (U) definition: one unit generates a decreasein turbidity of 0.001 OD/min at 520-540 nm of anEscherichia coli K 12 dried cell suspension (ini-

tial optical density 0.6) in 1.0 mL of 0.004 M

phosphate buffer, pH 7.2 at 30 °C.

Biochemical indices

The biochemical indices of animal blood andsera: hemoglobin level, red blood cell (RBC)count, white blood cell (WBC) count, leukocytedistribution, protein (total and electrophoreticallysubdivided components), glucose, calcium, andphosphorus levels, alkaline phosphatase and amy-lase activities were determined with the help ofwell-known and widely applied methods of vet-erinary laboratory medicine summarized in twohandbooks (Belyakov, 1975; Smirnov et al, 1 98 1 ).The bactericidal activity of the blood sera wasdetermined turbidirnetrically according to theprocedures described in Matusevich et al (1971 )and the number of sulfhydryl groups was deter-mined by amperometric titration (Kedrova, 1962). ).

Post-mortem examination

At the end of the experiments (30 days) 24 hafter the last dose of the preparation was ingested,three rabbits in each of the four groups and sixcalves in each of the two groups were killed for

post-mortem examination. The same number ofrabbits and calves were killed for this purposeagain IS days later. Post-mortem examinationincluded macroscopic observations and

histopathological examination. Different seg-ments of the gastrointestinal tract (rumen andabomasum in calves and stomach in rabbits, duo-denum, jejunum and ileum) as well as the brain,heart, kidneys, liver, lungs, lymph nodes(mandibular and iiiesenteric), pancreas, spleenand spinal cord were used for preparing histo-logical specimens. Post-mortem examination ofall animals was performed by board-certifiedpathologists.

Veterinary-sanitary examination of the rab-bit and calf meat and the associated laboratorytests were performed according to the regulatoryguidelines for meat inspection (anonymous,1986) approved by FSU Chief VeterinaryMedicine Board of Ministry of Agriculture.

Statistical evaluation

Each index was determined at least three times.The results are reported as mean t SEM. Mean

values in the text and the tables were comparedusing the Student t test, ANOVA test as well asmethodological references to biological statis-tics (Rokitskii, 1973). A P < 0.05 was consid-ered significant.

RESULTS

Mouse experiments

When lysosubtilin was administered as asingle dose per os to mice in doses reach-ing up to 2 x 1 0&dquo; U/kg of weight, no signof toxicosis was observed. When injectedinto the subcutaneous tissue of white mice,the LD50 was equal to 5 x 105 U/kg ofweight. The latter result was calculated frommortality data, which were as follows. Therewere no deaths either among the control ani-mals or in the group where the lowest dose

of lysosubtilin (5 x 104 U/kg of weight) hadbeen applied. The mortality rate (numberof animals/percentage) in other groups was:3/25, 10/83.3, 11/91.7, 12/100, lysosubtilindoses being 2, 7.5, 15 and 50 x I OS U/kg ofweight, respectively.When lysosubtilin was administered in

doses of 2, 8 and 20 x 104 U/kg of weighttwice a day for 30 days, no experimentalanimals died. The animals continuously

gained weight in both the study and controlgroups (table I). Weight changes were con-sidered to be toxicologically non-signifi-cant, as they were too small and/or showedno dose relationship. No negative impactwas observed later either. Thirty days later(60 days after the start of the experiment)all mice remained alive. No changes in theirbehaviour were observed.

Rabbit experiments

The experiment showed that when admin-istered in doses of 2, 8 and 20 x 104 U/kg ofweight twice a day for 30 days lysosubtilindid not cause any evident sign of clinicaltoxicosis. The animals from all groupsgained weight at almost the same rate(table I).

The effect of lysosubtilin, when appliedin doses of 8 and 20 x 104 U/kg of weighttwice a day for 30 days, on rabbits’ bloodand sera indices are shown in table II. All theindices in both the control and study groupsfluctuated within a normal range as shown intable II, but the ANOVA test revealed sta-

tistically significant differences in

hemoglobin, WBC, protein, and glucoselevels between the groups. It was determinedthat the above mentioned doses in rabbits

did not cause any significant changes in theWBC percentage ratio (data not shown).Blood stain smear examination did notdemonstrate the phenomena of either aniso-cytosis or poikilocytosis in the RBC. Norwere any pathological changes in the cyto-plasm and nuclei of WBC recorded.

During the post-mortem examination ofrabbits slaughtered I and 15 days after thelast dose of the preparation was fed, nochanges in the brain, spinal cord, mucosalsurface of stomach and intestines, heart, kid-neys, liver, lungs, lymph nodes, pancreas,and spleen, in comparison with the controlgroup, were observed.

Veterinary-sanitary evaluation of theslaughtered rabbits’ meat is discussed belowtogether with the experimental calves’ meatevaluation.

Calf experiments

Clinical observations of the study group ofcalves fed with lysosubtilin at a dose of4 x 104 U/kg of weight twice a day for 30days did not indicate any deviation fromnormal for the musculoskeletal, cardiovas-cular, respiratory, digestive, nervous andurinary systems. On days 2 to 10 of feed-ing lysosubtilin, an increased appetite wasobserved in the calves.

One of the most important factors of nor-mal development is weight gain. Theweights of the calves are given in table III.

This table shows that the experimentalcalves gained every day approximately100 g more than the control calves, the dif-ference being highly statistically significant(P < 0.001 ).

Some blood and sera indices of the calvesare presented in table IV. It shows that allindices in both the control and study groupswere within normal range and the changeswere statistically insignificant. As forchanges in the leukocyte formulation of thestudy group calves’ blood (data not shown),no abnormalities caused by lysosubtilin werenoticed. The microscopic examination ofblood smears showed no pathologicalchanges in WBC cytoplasm and nuclei. Noanisocytosis or poikilocytosis in RBC wereobserved either.

The results of the biochemical testsshowed that no negative changes occurred inthe status of the organs and organ systems ofthe body, which are most sensitive to toxi-cosis, due to the effects of the tested doses oflysosubtilin.

During post-mortem examination ofcalves slaughtered I and 15 days after lyso-subtilin was last fed, no deviations wereobserved from the normal status of themucosal surface of the rumen, abomasum,intestines (duodenum, jejunum and ileum),lymph nodes, brain, heart, kidneys, liver,lungs, pancreas, spleen, and spinal cord.

Veterinary-sanitary examination of theslaughtered calves and rabbits and laboratorytests of their meat showed normal commer-

cial and sanitary qualities. It was qualitymeat with no unusual smell or taste. As theanimals (rabbits and calves) were sl<zugh-tered I day after being fed with the prepa-ration for the last time, the conclusion wasmade that the slaughtered animals’ meatwas fit for human consumption 24 h afterthe final administration of lysosubtilin.

DISCUSSION

Until recently, strategies and tests used forevaluating the safety of newly proposeddrugs varied widely not only among differ-ent countries (Dooley, 1979; anonymous.1985, )99);Weingandetai, ) 1992), but evenamong organizations within one country asshown in an example from USA (Kinter etal, 1994; Murphy et al, 1995). Only recentlyhave initial recommendations from an inter-national scientific committee for animal tox-

icity and safety studies based on currenttechnical and scientific information been

published. According to the authors (Wein-gand et al, 1996) &dquo;it is hoped that these rec-ommendations will serve as the basis forinternational harmonization of clinical

pathology testing guidelines developed bygovernment regulatory agencies and pro-fessional standards organizations&dquo;.

The basis of our studies were the regu-latory guidelines of FSU Ministry of HealthCare provided for the evaluation of newenzyme drugs (anonymous, 1985). Generalinformation about lysosubtilin includingaspects of its manufacture, mechanisms ofaction, physico-chemical properties, com-plex structure, in vitro activity on differentbiosubstrates as well as a description of itspreclinical therapeutic and prophylactic effi-cacy study results on target species of ani-mals (all this is foreseen in the regulatorydocument) has already been described (Biz-iulevichius et al, 1987, 1989, 1995; Biz-iulevichius and Arestov, 1997). The sub-sequent indispensable information, foreseenin the document mentioned, on lysosubtilin

toxicology and safety data has never beenpublished previously with one slight excep-tion. Znaidauskas et al ( 1989) showed thatthe dose of 2 x 10!’ U of lysosubtilin dis-solved in water is non-toxic when applied tothe cow uterus. The current paper fills upthe gap.

Some general conclusions from our stud-ies regarding animals of the three speciesare as follows. No deaths and no treatment-related clinical signs were observed duringexperimental procedures cawied out per os.The body weight gain shown by the studyanimals was similar to that of normaluntreated animals of the same age andstrain/breed (table 1), except for the new-born calves, where a significant increase inbody weight gain influenced by lysosubtilinwas observed (table III). Macroscopic andhistopathological post-mortem examinationof animals at slaughter did not reveal abnor-malities.

The following are some explanationsconcerning our observations in one of theanimal species. It was shown that the feed-ing of lysosubtilin at dietary levels up to2 x 10!’ U/kg of weight to white mice didnot induce noticeable signs of toxicity. Theno-observed-adverse-effect-level of lyso-subtilin (NOAEL) in toxicity study wastherefore 2 x 10h U or 2 g/kg of weightwhich allows this product to be classifiedas non-toxic (Pariza and Foster, 1983).

The increase in WBC numbers (as well as

hemoglobin) in the blood of the study groupcalves in comparison with control ones(table IV) did not exceed the limits of thenormal physiological range (Fraser, 1991 ).The level of alkaline phosphatase activity,one of essential indices of hepatobiliaryevaluation in newborn calves (Pearson etal, 1995) and the results of histopathologicalexamination of the liver and spleen, organsassociated with blood cell formation or

sequestration also supported the non-toxi-city of lysosubtilin. On the other hand theincreased bactericidal activity in blood sera

on day 20 and 30 as well as the increasedbody weight gain in these calves confirmedour previous statements (Biziulevichius andArestov, 1997) on the positive impact oflysosubtilin on the development of an animaland its protective functions. It may be addedthat veterinarians themselves specializingin calf management were involved in the

experiments of evaluating lysosubtilin as adrug and they were highly appreciative(Prater et al, 1996).

Enzymes (lysosubtilin complex as well)are hiocatalysts of protein origin needed inonly minute quantities and will therefore bemetabolized when used per os during normalprotein digestion (Pariza and Foster,1983).This digestion will split the enzymes toamino acids which cannot be differentiatedfrom naturally occurring amino acids in ani-mal feed. Therefore, it is unlikely thatenzyme residues will be detectable in theanimal products. This speculation is con-firmed by a previous publication (Sirvydis etal, 1989), which showed that bacteriallysozyme, a preparation similar to lysosub-tilin, when used as feed additive to broilerchicks, did not accumulate in the internalorgans and muscles as determined with the

help of a radioactive iodine-12-5 labeledenzyme preparation.

Lysosubtilin has been used on a largescale in FSU for prophylaxis and treatmentof gastrointestinal disorders in newborncalves for some years. Data collected during gpostmarketing surveillance of lysosubtilinconfirmed the safety of this veterinary drug,and this is supported by the present results.

ACKNOWLEDGMENTS

We thank the veterinarians and techniciansinvolved. Our special thanks extend to

Dr M Liakhovich for his invaluable contributionin introducing lysosubtilin into veterinary prac-tice. The corresponding author is grateful to hisclass-mate K Kachkuviene for helping to pre-pare the English version of the manuscript. The

anonymous reviewer is appreciated for the crit-ical revision of the manuscript.

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