SORENESS

FIELD OF THE INVENTIO

[FsraOCHMj The invention in general relates to enxymes. More specifically the invention re a es to use o t e di est ve erayraes for the alleviating the sym toms of delayed on set tnuscle soreness (DQMS). ACKGROUN Of THE INVENTION

DESCRIP ION OF PRIOR ART

asite] Delayed oil et muscle soreness (POMS) is an exercise induced phenoraeaon and is a recurrent form, of s iffs ituury. it is preseated as a sensation of discomfort, pr om nantly with the skeletal muscles following unaccustome physical activity. Associated symptoms include muscle slmttening, increased passive s iffness, swelling, decreases in. strength and power, localized soreness and disturbed proprioception. This may even cause structural damage to muscle and connective tissue leading to alterations in the muscle function and joiat mechanics impairing the performance of athletes and. sportsmen. Fara0M3] Treatment strategies tor DOMS, both prophyl ctieally and i erapentically, ate well known in d e art, which include eryodterapy, stretching, and Anil atnnistory, drugs, idtrasound electrical current techniques, homeopathy; massage, compression, hyperbaric oxygen and exercise (Cheung si. ah, Delayed Onset Muscle Soreness Treatment Strategies and Performance Factors, Sports Medicine 2003: 33 (2): 145-164). Supplementation in the form of branched, chain amino acids (Shiaionmra et al, Braached-Chaia Amino Acid Supplementation Before Squat Exercise and Oelayed-Ouset Muscle Soreness, International lo rnai. of Sport HntritiDn and Exercise Metabolism, 2010; 20:236-244} and vitamin C (Connolly et ah. The effects of Vi amin€ su ple e tation on: symptoms of delayed onset rnuscie soreness, Journal of Sports Medicine and Physical Fitness, 2006; 46;462-467) are also administered as treatment for DOMS. Beck et ah (Effects of A Protease Suppleiaeal On Eccentric Exercise-Induced Markers Of DelayeoSOnset Muscle Soreness And Muscle Damage, journal of Strength and Conditioning Research, 2607; 2l(3);66hA¾?) tested, the validity of a protease based supplement o» the marke s of BOMS and muscle damage. However, absor tion issues sod the desitirction of enzym in the gut could limit the effectiveness of traditional anti~DOM5 enzyme (protease) therap . Also, t e efficacy of these treatments is limited arid- mc msistsnt o kg to the lack of aoderstamiiag siui-ounding the exact mechaaism of DOMS. The available treatment modalities are -useful in improving some symptoms of DOMS, but aot all symptoms are addressed.

[ParaWCMj It is the principle objective of the present inventios to disclose the perforrnas.ee of a comprehensive digestive enz me blend - DigeZynut® (Protease, Amylase, La tase, Lipase, Cel dase) as a therapeutic supplement for alleviating the symptoms of DOMS.

[Par iWSf The present mvention fulfils the aforesaid objective d also provides other related advantages,

SUMMARY OF THE I VE TIO

P&r&OOOS] The present invention discloses a combination of digestive: enzymes (Protease,. Amylase, Lactase, Lipase, Ceikriase) - DigeZymei ⁾ for the therapeutic management of delayed onset muscle soreness (DOMS) P¾ ¾eee?j Other f atures and advantages of the present invention will become apparent from the follow ng more detailed description, taken in co?rjnrsction with the accompanying images, which illustrate, by way of example, the principle of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS IParaSeOSJ ig J. shows the graphical representation of rneao and held dynamometer readings of rest subjects co rsummg DigeZyme® ar*d placebo timing Visits 0«3,

Pa C¾#lij ig.2 shows the graphical representation of me Algomeier readings in kg/enf ^; from the thigh muscle of test subj ects consuming DigeZyme® and placebo during Visits 0-3. jjPar¾08:i If Ftg-3 shows the graphical representation of mean A!gometer readings In kg/cm* ire-m the calf muscle of test subjects consuming DigeZymeii and placebo during Visits 0-3. Fara0M2 f¾4 is the graphical representation of the mean value of total time taken for Illinois Agility agility run test by test subjects consuming DigeZymel ^; and placebo during Visits 0-3. p¼raiK113f i¾g,S is ike graphical re esestatioa of mean value of McGili pain questimnaire t st vaiu « i m the tes subjects consuming DlgeZynie® am! placebo stmpkments daring Visits 0-3.

fPara(Rf!4| fig, 6 is the graphical representation of mean serum creatinine kinase levels of the test subjects commmag E geZyme® and placebo daring Visits 0-3..

fP&r&ClM $f fig,? s die graphical representation of mean serum lactate dehydrogenase levels of toe test subjects cQ.»$«Kii»g DigeZ e3& -and placebo during Visits 0-1,

BESCRIf ttC O THE MOST KEF1RRED EMBODIMENTS § a #16 In the most preferred embodiment, the present invention discloses a method for the therape tic managemen of delayed: onset muscle soreness nsing a comprehensive digestive enzyme blend of Protease,. Amylase, Lactase, lipase and Celkiase CDige£yrne®) _s said method, comprising steps of administering effective on ntrati ns of me enzymes to mammals presenting with symptoms of delayed onset muscle soreness. In a -related embodiment, the Individual enzymes in the digestive blend are present in me following concentrations: a) amylase: 24000 00% b) Ce!lokse: t 100 Cb% c) lipase: 200 FlP/g, d) Lactase: 4000 ALli/g and e Protease: 6000 PC/g, In another related em odiment, the symptoms of delayed onset muscle soreness Include pahy tenderness, inflammatlan, muscle damage and flexibility,

[f&rat!OJ.?! In another preferred embodlement, the invention, discloses a composition comprising a comprehensive digestive enzyme blend of Protease, Amylase, Lactase, Lipase and Cedukse iDlge¾mofO) tor use In die peude management of Delayed Onset Muscle Soreness (IX)MSf fParsCMtl& The specific examples included herein below illustrate me aforesaid, most preferred embodiments of the present Invention. fParaW f EXAMPLE 1: Study Besign ParaOO201 Prndhret description; DigeZynie* is an ⁱ off - white to creamy white powde of mithi -enzyme .com lex' ⁵ . This Multi -enzyme complex consist of amylase, protease, lipase, cellukse and lactase. Placebo capsnles containing equivalent weight of mahodextria. No di-iierenees in colour, taste ₅ exture or packaging were detectable between die two roducts. Capsules were- se led in IdentieallyHppearing, high-density polyethylene bottles with desiccani The composition of the .muf i-enzyme com ex is iven in Table L jPara8i2if TaMe 1. Com osi ion detail of niuitbenzyme complex (EiigeZyme) Formulation

S. No. ENZYME Enzynie activity (IJnits/g)

. Alpha-ao lase 24000 PU/g (Dextemking Unit /gram)

2 Ce!fuiase 1 100 CU/g (Cellu!ase Unit /gram)

3 lipase 200 F!P/ (Federationlnteritatm

4 Lactase 4000 A.Lli/g (Acid Lactase Obit/gram}

5 Neutral Protease 6000 P€/g (Protease Un on L-tyrosine basss gram)

Enaynie nnl s w»e defined as per Food Chemical Code (FCC), 5 ^ia eel. 2004,Xhe [National. Academy Press Washington, D.C, FarafI0221 E ks approval; Ethic approval was obtained from Sparsh Hospital for Advanced Surgeries, #146, mfttmry Road, Bangalore (registered under central drugs standard coairoi organization as per the gazette notification maobsr F .28- 10/45-1:1( 11 dated 21. Dec 1945 and last amended, vide notification number G.S.R. 76(E) dated 08 FEB 2012.) prior to tie initiatio of the- stud , j¾r&0823] Informed coaseat; A total of 20 healthy volunteers were kc ed in the study. A written . informal consent was obtained from the volunteers dining a _: scheduled initial screening visit. Adequate oral and written information concerning the study was, provided to the subjects and subjects were provided wi ample opportunity in consider their participation in the study. Alter obtainin a signed informed consen subjects were screened, for the study, jF»ra0<*24) Study designs For the DOMS model under study, a prospective, double blind, randomized, and placebo controlled design feature was selected. The analgesic efficacy of igeZyme¾as compared with a matching placebo. The pre specified efficacy end points of the study were assessed on D y 0 (pre exercise) and 72 hours post exercise. |Fsra002S| ar clpa s? Twent healthy males t no kaowa mt settloskeletal ^' patfeo gy participated m the study. Subjects w re exeloded if they met one or mo e of the following exclusion criteria: treatment with anty«.fiai«m8t i- ½>algesk «tk^id8nt drugs in the previous month, abnormal liver or renal foacti a tests, laboratory fedmg suggestive of an active i«flammatoty or infections process and presence of any kaow«: disease. The participants also completed a heahb history questionnaire es ned to idendfy die degree of risk t¾r cardiovascular or orthopaedic oonmlleadons during exercise. l¾ere were no withdrawals during the course of the study. ¾r¾W2hj Su ple men ati n with m Ui en¾ysite eoni fe: All participants in the experimental group received the Digezyme multi e me complex supplenientatioo over a 3 day period. During this period, they consumed one capsule ofrnnlrl enzyme complex thrice a day The participants is the placebo group received capsules of similar size and colour . They were given identical instructions on dosage of study supplement to be followed. Study supplements were prepared, and. provided by Sami Labs Limited, Bangalore. The administration of tr atment and placebo was blinded for both the participants and die investigator, There were no side effects reported by the participants as a, resu t of the supplementation,

|P¾r»882?J Data collection protocol: Subjects visited the elide on day 0 (baseline v sn) and subsequent visits on day i, day 2 and day 3 After recording vital signs (blood pressure, pulse rate, heart rate and respiratory rate), medical and medication history, physical examination was conducted. ff¾r¾§fl28| a»«i»i»¼atH>ft and Altoeatfen eoncealnsent;: The randorolsabon se uence was prepared by a Independent statistician, independent of the sponsoring organization and not invol ved in conduct r reporting of the study. An alpha numeric code was generated for both the active and placebo to improve the blindness of the study and concealment of allocations. Computer generated random allocation software (version 2,0) was used for the allocation of concealment. Block r¾ndomization (only one block) was followed wherein the subjects were randomized to receive either active or placebo. The randomisation codes were kept strictly confidential and were accessible only to authorized persons on an emergenc basis as per the Sponsor standard, operating procedures until the time of unblinding. ¾m0 2^1 Bitefilag: The smdy was double bl¾ded wherein neither the investigator nor the trial participants knew whether they would receive the active or the placebo. The investigational products were provided is pre labelled containers to avoid bias.

{$¾r»083$| EXAMPLE ΙΙί Procedures Followed; F¾r¾l li3l| Pr tocol f»r Istdaelng BOMS: Subjects were instructed to ingest the study s pplement am! report to the site after 24dioars for the baseline readings. After 10 hoars i¼st % heart rate and ratings of percei ved exertioa were monitored at test, every 5 mlaate during exercise and 10 minutes iato recovery. The parbcipants mounted a level motorized treadmill aod armed i for 5 miaut.es at a self-selected pace. Post S-min warm up, treadmill speed was increased until a heart rate of 80% of predicted taaxlaial heart rate was achieved aad were instructed to aiaintaia ib s pace for 5 mm. The treadmill grade at this time was adjasied to 10% and was aia ramed for 30 miaates. Subjects ohen completed a 5 minutes active coobdowa at a self-selected pace and a 5 minutes seated passive recovery period. Subjects were restricted from, indulging i» any other physical activity 24 hours prior aad 72 boars after the exercise session.

|Fair¾0i32 QuatsnTfyisg lauscle soreness using a!goiaefer: The h!nsele Soreaess Questionnaires (MSQ) required articipate to rate their geaera! soreness on a. scale of I (aorma! to 10 (very, very sore) for the right front thigh and right back thigh (Miller et a!..,. The effects of protease supplementation on skeletal muscle faaction aad DOMS following downhill running. Journal, of Sports Sciences 2004; 22;365~3?2). An algometer was used to quantify muscle pain by ap lying direct pressure ove the muscle. It Involves documenting the threshold at which the applied pressure over the masele is percei ed as a sensation of pain rather than a pressure; this is referred, to as the pressure paia threshold (PFT). The PPT has been demonstrated to be reliable for measuring pain threshol . The algometer standa d is to increase pressure linearly to 5 kg/crsT over 5 seconds according to the method recommended by Fischer. The la trument bas a I cm ^* rubber footplate aad a scale marked fro . 2 to 20 kg/ear\ in increments of 0.2 kg/em . Subjects were instructed to report as soon as the sensation of pressure changes to pain by sa ing 'paiuT and Ί will stop The footplate of the algorneter wa held perpendicular to the rauscle belly with the gauge turned away horn the subject and. the examiner. Pressure was increased at a rate o approximately I kg/eoxTs until the subject reported " ais The examiner then released the pressure aid lifted the algorneter off the muscle to read the gau e and record the measuremen The needle o the gauge as returned to baseline before each trial using the pressure release button on the algonietet,

jP&r&0O33) fur muscul r strength aitt! power »sl«g hm held dyna o meters A Band- Held Dynanxuaeter (HHD) was used, to quantify changes In muscle strength by oreasoring strength durmg maximal voluntary isometric contraction (MVC) of the muscle (Keiln et al, Hand-Held Dynaniontetry: Reliability of Lo e Extremity Muscle Testing In Healthy, Physically Active, Young Acklts, Journal of Sport Reh bilitation, 2008: l?; !bChl70). Each participant was asked to perform, three repetitions of maximal fco.ee extension and flex loo. contractions (MVC) at tbree different positions of kuee range of morion (0 degrees, 00 degrees, 120 degrees). The test angles were chosen to account for changes in muscle strength that could occur due to alteration, in the length of the rnosele at different positions of the kuee, fPa.ra.0O34j Illinois Agility uss Test; Agility was m.easra¾d using Illinoi agility run test (Geieheii B. Physical Fitness; A Way of life, 2nd ed. New York; John Whey and Sons, inc..,. 1979). The tests were conducted on a course 10 meters in length and 5 meters, in width. Agility time was recorded usin a stopwatch. The start, finish and. the two turnin points were marked with four cones and another four cones were placed down the center equal distance spaced 3,3 meters apart. Subjects were made to lie on their front (head to the start line) and hands by their shoulders. The rim. started from a standing start on the command ^l kd the stopwatch was started and the subjects were instructed to get up as quickly as possible and run around the course in the direction indicated, without knocking the cones o , to the finish line, at which the timer was stopped, ¾ aU 3S| Oeteome measures: MeGill pain questionnaire comprising of . 0 suhscales from 0 (no pain) to ID (worst possible pain) wa used to assess pais, (Mekack E. The MeOill Pais Questionnaire; Major properties and scoring methods. Pain.1075; 1:277-299; Sullivan et si, An experimental investigation of the relation between eatastrophizing and activit intolerance. Fain 2002; IO0;47~53). An algorneter was sed to experimentally induce pain on a predefined point on the patellar tendon fiv centimetres above the center of the patella, tenderness was assessed. Subjects ranked their pain perception on a scale from 0 to 10, On day assessments were takes at baseline (pre-exercise), post-exercise; and further, at 24, 4:8 and 12 hours post~esetci.se for each arm of the study. Secondary outcomes e!uded assessments of inflammation, muscle damage, flexibility, d the amount of energy expended prior to exercise.Biomarkers creatine kinase and lactate dehydrogenase were monitored pre exercise and 72 hours post exercise. Safety profile of the supplement was assessed by routine . ematology kidney and liver Inaction tests. Adverse events were monitored thro ghout the s udy.

[PsraOS ] Statistksl analysis: All testing was done using Statistical Analysis Software (SAS) Yersioa 9.2, All analyses was conducted osiug the intent-to-tteat population. Patients with no da a recorded for a particular parameter were automatically exeioded from the analyses of th t parameter, f r all the data set variables analysis of covariance (ANCOVA) and Wilcox, on signed rank stun test were osed aad. the level of significance was set as F < 0.03,

|Ρ»ηήβ37| EXAMPLE lU ResaMst

|Par¾O038| Physical dfcsracterfoties: ^' The population was essentiall ^' healthy without significant concomitant disease or medication intake. Screening characteristics of participants are presented in Table 2.

MearifSD) 59.90 (4,63) 1 59.68 (4,82)

W ste ¼r¾§M#l There ere BO atis k-ally slgnlikarst differences Between subjects in the placebo (E ^;;:: 10) nil the DigeZyme* ^' (a ^:::: 10} group. 0» the day of screemng.. the mean, weight of all the enrolled, subjects was 5.9.3 ±· 4,64kgs; em helghi was 163.8 ± 4.99 cnt and the mean BMI was 22.2 ± 1.5 Kg m ² . Par¾i¾Mij Efficacy E-val«¾fiai$r Delayed Onset Muscle Soreness-quality of life was analyzed throughout the study period as primary efficacy measure. The *p ^* value suggests that there was a statistically significant change in these symptoms from baseline to final visits, between, the placebo and active arms, Statistical analysis using Analysis of Co- Variance CANCOYA) showed the primary efficacy parameters were statistically- significant (p<0.O5) between the nmutienzvnie complex and placebo groups (Table 3),

*p mlt significant (<0J)5) heiwem the n-e&immf groups. |Fsraee43 furthermo , comparative mesa values of efficacy assessments between. Mali! enzy ae complex and. placebo _. groups ^' across various visits (baseline, day I, clay 2 and day 3) are presented .for efficacy parameters (Figs. !»?)« Patients who were on Mold enxyme complex bad statistically significant difference for the effic cy parameters on day 3 when compared with placebo.

|Far¾0844| Pair* ime m nt MeGili pai uest onna e is a multidimensional pain instrument, the final score is a sum of all he ten individual p n questions. The first a e McGil! pain rniestionnaires were based on assessment of pain (current pain, least pain, arid worst pain) while the ' ent , on the degree of np.mhn.ess and its Interference with function. When differences between mute enzyme complex and placebo were comp re , the pans score showed high statistical significance (p-O.O dl ),

|Fa »0e S| Te»d«raess a ses ments On. fire tenderness qti tient, subjects taking m»Iti eaxyme complex demonstrated significantly less teademess, 72 hours after exercise (p ^™; 0.042 Ϊ. ^ a MMhf Musc e Pam g Assessments?: .Liberation of biochemical substances such as creatine kinase, lactate dehydrogenase, protein metabolites and. myoglobin occurs from muscle cells approximately 24 .hours- post exercise and have been found in plasma up to 48 ho ars. Creatine kinase hemg a surrogate index of .muscle d amages m ore indicative of damage or gaps in the sarcotemtna, The CK response was less in the multi. enxyme complex group suggesting the rnernhrane Integrity was rnaintaiBed to greater extent than the placebo group (Fig, t>>. 24 hours post exercise, subjects on the placebo group showed trend towards a higher level of CK. than foe multi etrrynie complex, group, Tfeis trend co imted throug the 72 boar assessment, Differeace between the multi esrayrne complex group and placebo m CK values was not statistically significant lactate dehydrogenase also shewed a sim lar phenomenon, which, trended higher at 24 and 72 hours post-exercise In the placebo group (Fig.7).

[ aradM?! Flexini? md Extessina Measurements: On analysing the pre exercise leg flexion measurements, they were found to he equal hetweem the groups for the left leg. Wbereas foe flexion, measurement was found to be significantly greater (p ^;;; 0.049) for the right leg in the placebo group, Wbea the post exercise flexion measurements were analysed, only the 244ΚΜΓ .right le flexio me wemer$t was found to be significant (ρ ^:~ 0,0ό4) k the txmlti enzyme com lex group.

|Para00 i Safety Evaluations.; Vital signs sucfe as Blood Pressure, Respiratory R e, False Rate aad my abnormal laboratory parameters were considered for safety evaluations. No clinically significant changes were recorded for descriptive physical examination in both the groups (Mold enzyme complex and. placebo). Th safety of multl enzyme complex was assessed, using adverse evetrt data (occurrence, intensity, and relationship to study drug),. No ad erse events were noticed in the -study,

{Pa.r¾0849J In tbe present -study, ie D g§Zyme€> multi-enzyme complex capsules demonstrated significant improvement in subjective pain and tenderness, with so significant im ro ement in le vels of markers of inflammation, muscle damage or muscle flexion,

|Parst 5§i While the invention bas been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in tbe art that tire mversiion is not limbed thereto, Ratber, the scope of the iBveation is to be interpreted only in. conjunction with the appended claims.