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Tetracycline and its modifications .pptx seminar 5

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Dr. Mitali Thamke

This document discusses tetracycline and its modifications. It begins with an introduction to tetracyclines as broad spectrum antibiotics discovered in 1948. It then covers the classification, structural activity relationship, and mechanism of action of tetracyclines. The rest of the document discusses the spectrum of activity, uses, side effects, drug interactions, resistance, and local drug delivery of tetracyclines for periodontal applications. It provides examples of commercially available tetracycline-containing local drug delivery products and studies on their effectiveness.

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TETRACYCLINE AND IT’S MODIFICATIONS PRESENTED BY : DR. MITALI . V. THAMKE I M.D.S
INDEX 1. Introduction and background 2. Classification 3. Structural Activity Relationship 4. Mechanism of action of tetracyclines 5. Spectram activity 6. Uses of tetracyclines
INDEX 7. SIDE EFFECT OF TETRACYCLINE 8. LOCAL DRUG DELIVERY 9. TETRACYCLINE AS A HOST MODULATING AGENT 10. CHEMICALLY MODIFIED TETRACYCLINE 11. CONCLUSION 12. REFERENCES
Definition: Tetracyclines are octahydro napthacene derivatives which are bacteriostatic and broad spectrum antibiotics that kills certain infection - causing microorganisms and are used to treat wide variety of infections.
The tetracyclines were first discovered in 1948 as fermentation product of golden colored soil bacterium named Streptomyces aureofacians. Chemical isolation and purification of its products produced the compound Chlortetracycline in 1948, the first tetracycline to be fully characterized both chemically and clinically. Its advantages were a larger spectrum of activity & was better-tolerated by & less toxic to some individuals. INTRODUCTION AND BACKGROUND:
According to duration of action: Short-acting (Half-life is 6-8 hrs) • Tetracycline Chlortetracycline Oxytetracycline Intermediate-acting (Half-life is ~12 hrs) Long-acting (Half-life is 16 hrs or more) • Doxycycline Minocycline Tigecycline • Demeclocycline Methacycline
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3rd generation (After 1972) 4th generation 1st generation (Until 1965) 2nd generation (1965-1972) Tetracycline; Chlortetecycline; Oxytetracycline; Demeclocycline; Glycylcycline Tigecycline Azatetracycline Alkylaminotetracyclin e Doxycycline; Lymecycline; Meclocycline; Methacycline; Minocycline; Rolitetracycline DEPENDING UPON THEIR SYNTHESIS
OH OH CONH2 OH R1 O O R2 R3 H R4 H N(CH3)2 OH ABCD 1 2 3 4 4a 5 5a 6 6a 7 8 9 10 10a 11 11a 12 12a N(CH3)2 Increases activity Conversion to nitriles causes a 20 f old increase in activity Modif ication leads to loss of activity =CH2 Increases the Antibacterial activity Elimination of 6-OH group increase lipophilicity & more stable to acids. Ex: Doxycycline. ‘D’ ring should be always aromatic Changes in this ring Leads to biological inactivation of the molecule. Additional glycyl amino substitution at the 9th Position leads to the new Class of antibiotics the glycylcyclines. EX: Tigecycline.(Tygacil) The keto-enol tatomerism Between c2 and c3 are very important f or biological activity. Inviolate zone is essential The linearly f used tetracyclic nucleus is most important f or the antibiotic activity. Electron donating (or) electron withdrawing groups at c7 increased Antibacterial activity Substitution with –OH Produce water soluble derivatives which can be administered orally. Epimerization at c4 and dehydration at 5a results loss of activity. 9 Structural Activity Relationship:
 Tetracyclines inhibit protein synthesis by binding to the bacterial ribosome involved in the translation(protein synthesis) process and making them bacteriostatic.  The bacterial ribosome is a 70s particle made up of 30s subunit and 50s subunit.  The 30s subunit binds mRNA and initiates the protein synthesis.  The 50s subunit combines with the 30s subunit-mRNA complex to form a ribisome then binds aminoacyl tRNA and catalyses the building of the protein chain..  There are two main binding sites for the tRNA molecule.  The peptidyl(p-site) binds the tRNA bearing the peptide chain  The acceptor aminoacyl site (A-site)  Tetracyclines reversibly bind to the 30S subunit at the A-site to prevent attachment of the amino acyl tRNA, terminating the translation process. . . 10
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ANIMATION ILLUSTRATING THE ROLE OF TETRACYCLINES IN BLOCKING TRANSLATION DURING BACTERIAL PROTEIN SYNTHESIS
ABSORBTION DISTRIBUTION EXCRETION Absorbed from duodenum and small intestine. Forms insoluble complex with Ca+,Mg+,Al+,Zn+. Absorption of tetracycline is impaired by the concurrent use of dairy products. Mechanism responsible for decreased absorption appears to be chelation of divalent and trivalent cations. Throughout the body Cross the placenta &enter the fetal circulation &amniotic fluid Kidney through glomerular filteration(except minocycline)
ROUTES OF ADMINISTRATION • Oral • Parentral • Topical
THERAPEUTIC USES I. Antibacterial properties II. Non antibacterial properties
 Gram +ve & -ve bacteria  Spirochetes  Mycoplasms  Rickettsiae  Candida Albicans  Mycoplasma Pneumoniae  Chlamydia Trachomatis  Borrelia Recurrentis  Yersinia Pestis  Vibrio Cholerae  Campylavacter Fetus  Brucella Specie  Streptococcus Pneumonia  Neisseerie Gonorrhoeae 16 • Tetracyclines are broad spectrum antibiotics. They are active against following micro organisms:
NONANTIBACTERIAL PROPERTIES • Anti-inflammatory effect • Immunosuppressive property • Suppression of antibody production in lymphocyte • Reduction of phagocytic function of PMN • Reduction of leukocyte & neutrophil chemotaxis • Inhibition of lipase and collagenase activity • Enhancer of gingival fibroblast cell attachment • Antitumor activity
ADVERSE EFFECTS 1. Teeth and bones : • Tetracycline chelates calcium, forming a tetracycline-orthophosphate complex and are deposited in area of calcification in bone and teeth • Administration in pregnant women • lead to yellow staining of teeth in infants; defective formation of enamel and hypoplasia of teeth may occur • Tetracycline deposit in foetal bones and may reduce linear growth • Also be avoided in infants and children upto age of 12 years
• Tetracycline can cause skin reaction. • Tetracycline get deposited in nails,which may cause nails to fluroscence. • Photosensitivity manifested by marked erythema. • Superinfection : supression of the normal intestinal microflora is liable to occur after prolonged tetracycline therapy.
• Mild nausea, vomiting, diarrhea. • White patches or sores inside your mouth or on your lips . • Swollen tongue, trouble swallowing. • Loss of appetite, • Jaundice (yellowing of the skin or eyes). 20
Drug Possible sequence Penicillin Antagonism of bactericidal action Antacids &iron preparation Impaired absorption of TCl insulin Oxytetracycline enhances hypoglycemic action Contraceptive pills Pill failure Cimetidine Reduced bioavailablity of TCl Digoxin  in serum digoxin concentration Lithium carbonate  serum level of lithium Warfarin sodium Enhanced anticoagulant effect Carbemazapine and phenytoin Reduced serum concentration of Doxycline DRUG INTERACTIONS
RESISTANCE TO TETRACYCLINE 3 MAIN RESISTANCE MECHANISM: • accumulation of tetracycline a)antibiotic influx b)energy dependent efflux pathway • Presence of ribosomal protection proteins • Enzymatic inactivation of tetracycline (Speer et al 1992)
• tet gene responsible for tetracycline resistance • tet (B)-coding for efflux • tet(M)-ribosomal protection protein • tet(X)-Enzymatic alteration
SYSTEMIC TCL THERAPY IN PERIODONTAL USE
CHRONIC & AGGRESSIVE PERIODONTITIS IN THE ADULT PATIENTS • Systemic Tetracycline –for 30 days produce  in clinical attachment level& in probing depths (Haffajee et al 1995) • Disadvantages • No statistically significant difference were measured in sub gingival composition between DOXY &placebo patient. • Recurrent disease activity
REFRACTORY PERIODONTITIS • Drug regimen • Doxycline for 3 week • Reduced relative risk for periodontal breakdown over a seven month period by 43% (Mc cullon et al 1990)
DISADVANTAGES • Recurrent disease activity (Van winkelhoft 1989) • Subgingival microorganism not sufficiently suppresed (Muculloh 1990) • No difference in subgingival microflora between placebo and DOXY treated (Kukarni et al 1991) •  in the number of organism (Muller 1990) • Super infection and opportunistic pathogens (Brogd et al 1985)
AGGRESSIVE LOCALISED PERIODONTITIS • Drug regimen • Tetracycline HCl(250mg four times daily for 14 days every 8weeks) or DOXY 100mg 2 tablets stat and then single tablet for14 days. • Periodontal surgery together with systemic Tcl-Hcl were able to decrease the periodontal AA below detectable level. (Slots &Rosling 1988) • ADVANTAGES • Reduced gingival inflammation • Gain in clinical attachment and alveolar bone (Genco et al )
DISADVANTAGES • Reactivation of disease (25%) (Lindhe 1982) • Periodontal destruction was noted with post treatment levels (Slots & Rosling 1993) • TCl did not suppress AA in all LJP
MOUTH RINSE SUBGINGIVAL IRRIGATION SYSTEMIC DELIEVERY CONTROLLED DELIVERY Reaches site of disease activity Poor Good Good Good Adequate drug concentration Good Good Fair Good Adequate duration of therapy Poor Poor Fair Good DIFFERENT DRUG DELIVERY SYSTEM
LOCAL DRUG DELIVERY
KORNMAN2 CLASSIFIED CONTROLLED RELEASE DELIVERY SYSTEMS IN PERIODONTICS BASED ON THE RELEASE OF THE DRUG INTO:RESERVOIR WITHOUT RATE CONTROLLING SYSTEM: RESERVOIR DEVICES WITH RATE CONTROLLING SYSTEM: Includes devices such as hollow fibers filled with a therapeutic agent in which the agent is released simply by diffusion through the reservoir wall. The most common forms include solvent action on coated drug particles, microporous polymer membranes or monolithic matrices, or erodable polymeric matrices. Classification based on the rate controlling system Kornman
2.RAMS & SLOTS (1996): DEPENDING ON THE USAGE PERSONALLY APPLIED (In patient home self-care) PROFESSIONALLY APPLIED(In dental office)
BASED ON DURATION OF ACTION (GREENSTEIN & TONETTI 2000) A) SUSTAINED RELEASE DEVICES Drug delivery for less than 24 hrs require multiple applications follow first order kinetics B) CONTROLLED DELIVERY DEVICES Duration of drug release exceeds 24hrs administered once follow zero order kinetics
INDICATIONS: CONTRA-INDICATIONS:  Periodontal patients with known hypersensitivity reaction to any of the antimicrobials used for periodontal therapy.  Patients susceptible to infective endocarditis are contraindicated for irrigation devices to avoid the risk of bacteremia.  Delivery of antimicrobial agents using ultrasonic scalers is contraindicated in asthmatics, infective conditions (AIDS, TB) and those with cardiac pacemakers  Isolated periodontal pockets (>5mm), with successful phase 1 therapy  Periodontal patients who are medically compromised where surgical therapy is contraindicative.  In combination with mechanical debridement or alone.  In-patients who are suffering from recurrent or refractory periodontitis.  During periodontal regenerative procedures.
• Advantanges: 1. Attains a 100- fold higher concentration of anti micro- bial agent in subgingival sites. 2. Reduces patient dose by over 400 fold thereby reduc- ing chances of drug resistance and side effects caused by systemic antibiotics 3. Small doses can be administered. 4. Maintain contact with the pathogens in the infected area for a prolonged period of time. 5. Maintains effective concentration. Goodson J (2000)
• Disadvantages: 1. Patient may not comply to placement of the drug sub- gingivally. 2. Diffculty in placing the device at the base of the pocket. 3. Lack of manual dexterity. 4. Does not have any effect on adjacent or nearby structures such as tonsils, buccal mucosa etc so may cause chances of re- infection. 5. Time consuming 6. Costly
PROBLEMS • Flushing action of GCF • Small volume of periodontal pocket • Pressure exerted by tonus of periodontal tissue • Drug reservoir in the periodontal pocket will be depleted within hours or days after placement
LOCAL DELIVERY DEVICES Tetracycline are available in following local delivery devices : 1. Fibers 2. Films 3. Gels 4. Strip 5. Microparticles 39
COMMERCIALLY AVAILABLE PRODUCTS 1. Tetracycline fibers (Actisite, Alza Corp., Mountain view, California) 2. Minocycline ointment (Dentomycine, Lederle, UK & Periocline, Sunstar, Japan) 4. Doxycycline hyclate in a resorbable polymer (Atridox, Atrix Labs, CO) 6. Minocycline microspheres (Arestin, Ora pharma, North Carolina, USA).
TETRACYCLINE –CONTAINING FIBERS(ACTISITE) • FDA approved • Non resorbable, biologically inert, safe plastic copolymer (ethylene &vinyl acetate) loaded with 25% w/w tetracycline hcl powder packed as thread of 0.5mm diameter &23cm length • It maintains constant concentrations of active drug in the crevicular fluid in excess of 1000 μg/mL for a period of 10 days.(Maurizio S etal) • In contrast GCF conc. of only 4-8 microgram/ml were reported after systemic administration, 250 mg qid for 10 days.
• Bioresorbable form is PERIODONTAL PLUS AB • Biodegrade within 7 days ,so no 2nd appointment • Gel (Maheshwari etal) • Tetracycline-Serratiopeptidase-Containing Periodontal Gel • Formulation has shown statistically significant results along with scaling and root planing.
• STUDIES • Singh et al.(2009)- 3 months - No difference in the results achieved with local tetracycline hydrochloride or local metronidazole as adjuncts to mechanotherapy. • However, both antibiotic therapies resulted in greater improvement in microbiological parameters when compared to mechano therapy alone.
• Sadaf et al.(2012)-Tetracycline fibers- 3 months • Higher reduction in plaque index, gingival index and in the clinical probing depths of the tested group than of the control group at all time intervals -15, 30, 60 and 90 days. • Tetracycline fiber therapy enhances the benefits of SRP in the treatment of chronic periodontitis. (Gupta Nidhi et al ,2015)
SUBGINGIVAL DOXYCYCLINE • Doxycycline is a bacteriostatic agent • Has the ability to down regulate MMP’s . • The only FDA approved 10% Doxycycline in a gel system ATRIDOX (42.5 mg Doxycycline) is a subgingival controlled-release product composed of a 2 syringe mixing system. It is the only local delivery system accepted by ADA
• Doxycycline levels in GCF peaked to 1,500 - 2000 μg/ml in 2 hours following treatment with ATRIDOX. • Local levels of Doxycycline have been found to remain well above the MIC for periodontal pathogens (6.0μg/ml) through Day 7. • 95% of the polymer is bio absorbed or expelled from the pocket naturally within 28 days.
• Locally applied controlled release DOX gel may partly counteract the negative effect of smoking on periodontal healing following no surgical therapy.(Tomasi C and Jan LW)
STUDIES  The FDA has also approved doxycycline hyclate in a bioabsorbable polymer gel as a stand-alone therapy for the reduction of probing depths, bleeding upon probing, and gain of clinical attachment (Kim TS etal,2002)  Deo et al.(2011)- 6 months - Doxycycline hyclate 10% as an adjunct to SRP provided significant reductions in PPD and gains in CAL compared to SRP alone.
SUBGINGIVAL MINOCYCLINE • A bacteriostatic antibiotic has been tried clinically via in three different modes i.e. film, microspheres, and ointment. Film: • Ethyl cellulose film containing 30% of Minocycline were tested as sustained release • complete eradication of pathogenic flora from the pocket after 14 days
Microsphere: • A new, locally delivered, sustained release form of minocycline microspheres (ARESTIN) for subgingival placement is available. • The 2% minocycline is encapsulated into bio-resorbable microspheres (20-60μm in diameter) in a gel carrier and has resorption time of 21 days. • Gingival crevicular fluid hydrolyses the polymer and releases minocycline for a period of 14 days or longer before resorbing completely.
Ointment: • 2% minocycline hydrochloride in a matrix of hydroxyethyl-cellulose, amino alkyl-methacrylate, triacetine & glycerine. • DENTOMYCIN –european union • PERIOCLINE -JAPAN • The concentration of minocycline in the periodontal pocket is about 1300μg/ml, 1 hr after single topical application of 0.05 ml ointment (1mg of minocycline) and is reduced to 90μg/ml after 7 hrs.
STUDIES • Timmerman et al (1996) reported that there was no benefit of employing 2% minocycline gel as an adjunct to SRP to reduce probing depths at deep sites • Steenberghe et al (1999) that combined therapy provided a better result than SRP alone at sites >7 mm deep. • Jung et al.( 2012) -Minocycline hydrochloride2% -Reductions in PPD, BOP and gain in CAL were significantly greater at the minocycline ointment in association with flap surgery site than at the flap surgery site alone
ISSUE SYSTEMIC ADMINISTRATION LOCAL ADMINISTRATION Drug distribution Wide distribution Narrow effective range Drug concentration Variable levels High dose at treated site,low levels elsewhere Therapeutic potential Reach widely Act better locally Problems Side effects Reinfection from non- treated site Clinical limitations Require good patient compliance Infection limited to treated site Diagnostic problem Identification of pathogens Identification of sites to be treated COMPARISON OF SYSTEMIC &LOCAL DRUG DELIVERY
HOST MODULATING THERAPEUTIC AGENTS • Sub Antimicrobial Dose of Doxycycline • Chemically modified Tetracyclines(CMT). • Synthetic –antiMMPs • Bisphosphonates. • NSAIDs-
LOW DOSE TETRACYCLINES • Golub et al recommended - 20 mgs Doxy capsules,twice a day for 2 weeks . • This low dose had no antimicrobial effect and hence did not lead to development of resistant microorganisms in sub gingival plaque.[Golub 1985,Thomas 1998,Walker et al 2000]. • However the anticollagenase activity was unaffected &significantly reduced collagenase activity in GCF was demontrated.[Golub 1990]. • Croat et al 1996 showed improved clinical parameters [attachment level , probing depth] when administered periodically over 6 month period.
SUB ANTIMICROBIAL DOSE OF DOXYCYCLINE • Doxycycline hyclate (Periostat): "Research is in My Blood" Dr. Lorne Golub, Developer of Periostat, received the ADA's Gold Medal Award for Excellence in Dental Research in 2006. • Available as 20-mg capsule, prescribed twice daily for use. • Approved by US Food and Drug Administrator (FDA), for the adjunctive treatment of periodontitis. • It acts by suppression of the activity of collagenase, particularly that produced by polymorphonuclear neutrophils. • It does not exhibit antimicrobial effects but can effectively lower MMP level
• Mcnamara et al attributed the lack of side effects to the 92% reduction in blood levels of the drug in patients on the low- dose doxycycline • Goldberg et al. found that Combination of SDD plus low-dose flurbiprofen, together with repeated bouts of nonsurgical periodontal therapy, produces improvements in nonresponsive patients (refractory periodontitis).
LIMITATION OF SDD • Either composition or resistant levels of microflora was unaltered. (walker et al 2000) • More aggressive forms of collagenolytic disease can’t be controlled.
ANTI-COLLAGENESE EFFECT • Inhibit active collagenase from the host • Prevention of conversion of pro-MMP to active MMP • Down regulation of pro-MMP expression • Excessive proteolysis of pro-MMP into enzymatically inactive fragments. • Prevent MMP’s from degrading/inactivating the serum protein 1 – proteinase inhibitor. (Golub et al 1990, 1994) • Collagenase produced from PMN (MMP-8)than from fibroblast (MMP-1).
CHEMICALLY MODIFIED TETRACYCLINES
The unexpected ability of tetracyclines to inhibit the breakdown of connective tissue & bone by a non-antimicrobial mechanism was first reported over 6 decades ago by McCormick 1957, Wilkinson, 1958, & more recently, produced & characterized by the group of McNamara, T. F., Golub, L. M., D'Angelo, G.,& Ramamurthy, 1986. Based on the thoroughly explored chemistry of the tetracyclines, a number of tetracycline analogs can be synthesized with side-chain deletions or, in some cases, moieties added to the parent tetracycline molecule.
Golub and co-workers 1983,84 made the seminal observation that TC’s inhibit collagenase activity and, thereby, launched the current interest in their non-antimicrobial properties. Their studies showed that: 1) Severely hyperglycemic, diabetic rats had reproducible, elevated levels of collagenase activity in gingiva (and skin); and 2) This increase in enzyme activity probably led to severe periodontal breakdown.
3) Furthermore, such an increase likely resulted, at least in part, from a shift to Gram-negative microflora in the subgingival plaque with more of its bacterial endotoxin concentrated in the gingival sulcus. Endotoxin penetration into the subepithelial connective tissue could then stimulate host cells; i.e., fibroblasts, macrophages, and keratinocytes to produce collagenase.
67 Golub and co-workers modified their experimental diabetes protocol using tetracycline therapy and germ-free rats to determine whether the Gram-negative microflora increased collagenase levels. The Diabetic Rat Model:
68 Conventional Rats Gnotobiotic Rats Periodontitis & Diabetes Minocycline 65% of pathologically excessive mammalian collagenase activity was reduced. Diabetes Minocycline 70% of diabetic gingival collagenase activity was reduced.
As a result of these initial studies, Golub et al. proposed: (1)That tetracyclines, but not other antibiotics, can inhibit collagenase by a mechanism not dependent on the drug's antibacterial efficacy & (2) That this newly discovered property of tetracyclines could provide a new approach to the treatment of diseases, such as periodontal disease, & also including other (medical) disorders like rheumatoid and osteoarthritis (as well as several cutaneous and other diseases) that involve collagen destruction.
70 Tetracyclines are known to inhibit collagenases (& some other, but not all, matrix metalloproteinases or MMPs from a variety of cells: Neutrophils, Macrophages, Osteoblasts, Chondrocytes, and A wide range of tissues: skin, gingiva, cornea, cartilage, and rheumatoid synovium.
Tetracyclines inhibit PMN but not fibroblas collagenase: PMN’s Collagenase 2-25µg/ml of Minocycline Inhibited Fibroblast Collagenase 250µg/ml Minocycline required Not Inhibited
Tetracyclines inhibit PMN but not fibroblast collagenase • It has been suggested that PMN’s provide the major source of collagenase that mediates tissue breakdown during inflammatory periodontal disease, • Fibroblasts contribute the collagenase required for connective tissue remodeling in normal gingiva. • Therapy with these drugs would be expected to reduce pathologically elevated collagenolytic activity (e.g., during inflammation), but not the collagen turnover required to maintain normal tissue integrity.
73 To identify the site of the anticollagenase property, Golub and co-workers 1991, synthesized 10 different analogs of tetracyclines known as chemically-modified tetracyclines (CMTs 1-10). All 10 analogs lacked antimicrobial efficacy and inhibited collagenase activity, but only 1 did not.
STRUCTURAL FORMULAE OF THE TETRACYCLINE OH O OH O O C NH2 OH N(CH3)2 HHOHCH3 OH 10 9 8 7 6 11 12 5 1 2 3 4 Site of antimicrobial action. Golub et al removed the dimethyl amino group from the C-4 atom and this Chemically modified tetracycline.[CMT].lost all its anti microbial property but retained its anticollagenase effect.
O OH OHCH3 OH OH O OH CONH2 CMT-1 [4-Dedimethylamino tetracycline] CMT-7 [12a-Deoxy-4-dedimethylamino tetracycline] O OH OHCH3 OH O OH CONH2 4 3 2 1 12 a
76 CMT-2 or tetracyclinonitrile was produced by dehydration of the carboxamide residue at carbon 2. CMT-3 - produced by removing the hydroxyl & methyl groups on carbon 6 & 4.
77 CMT- 4 - produced by removal of dimethylamino group from carbon 4 of chlortetracycline. when the carbonyl oxygen at carbon 11 and hydroxyl group at carbon 12 were removed by converting tetracycline to the pyrazole derivative or CMT-5, as described by Valcavi et al., the collagenase- inhibitory activity of the molecule was lost.
CMT-5-TETRACYCLINE PYRAZOLE O OH N(CH3)2 OHCH3 OH OH N N CONH2 CMT-5 (the pyrazole analogue of TC) (Valcavi et al,1963), which lacks the Ca2+ and Zn2+ binding site at carbon-11 and carbon-12 of the TC molecule,does not inhibit MMP activity, but it retains its ability to prevent pro-collagenase activity by HOCL inhibition. ( ROS-reactive oxygen species) - Sorsa et al 1998
CMT-8 ,the non-antimicrobial analog of doxycycline, appears to increase the MMP-inhibitory efficacy ,matrix sparing activities. (Paemen et al,1996; Zernicke et al,1997; Greenwald et al, 1998; Ohyori et al 1998) CMT8>CMT3>CMT1 O OH OHCH3 OH OH OH CONH2 O CMT-8 [6a-deoxy,5hydroxy,4-Dedimethylamino doxycycline]
80 Mechanism of action of CMTs: The proposed mechanism of action of CMT’s results from their ability to bind metal ions, particularly Ca2+ and Zn2+, which are required by enzymes to maintain its proper conformation and hydrolytic activity. Inhibition of active or pro-MMP could occur due to chelation of Zn2+ ions at the binding site in the catalytic domain, resulting in disruption of normal conformation of protein structure, & leaving it non-functional due to excessive degradation into small molecular weight, enzymatically inactive fragments.
81 CMTs have also been shown to downregulate expression of gelatinases, & thus to reduce the production of pro- enzyme (MMP-2 and MMP-9) (Golub et al., 1991, 1998). Also, CMTs may inhibit the activation of collagenases (MMP-1, MMP-8, and MMP-13), and Inhibit Stromelysins (MMP-3, MMP-10,and MMP-11) and Inhibit MT-MMP’s,
82 CMT’S
83 CMT’S
PMN’s Osteoblasts Procollagenase Collagenase ROS e.g. HOCL by neutrophils Secretes Activated CMT-1 100-400 µm Inhibits 40 – 80% TC effects on pro- collagenase activation:
Other mechanisms that have been proposed include: Inhibition of oxidative activation and increase in degradation of pro- MMP's, Inhibition of production of secretary non-pancreatic phospholipase A2, Inhibition of cytokine production, i.e., of TNF-alpha and IL-8, and Reduction of the expression of serine proteinase and trypsinogen-2. (Pruzanski et al., 1998; Kirkwood et al., 1999). Inhibition of protein glycation, and Inhibition of non-collagenolytic proteases. Inhibit secretion of other collagenolytic enzymes like Lysosomal cathepsin.
86 Bone resorption Inhibition: As examples of in vitro efficacy, TCs and CMTs were found to inhibit bone resorption in both organ and cell culture, regardless of whether the resorption was induced by parathyroid hormone (PTH), PGE2, or bacterial endotoxin (Golub et al, 1984; Gomes et al, 1984; Rifkin et al, 1994). CMT-1 and -3 and CMTs-6, -7, and -8 were effective inhibitors of bone resorption in culture (CMT-8 was the single most potent compound), whereas CMT-2, -4, and -5 were not.
87 Tc’s affect several parameters of Osteoclast function: Effects on Osteoclast function Diminish acid production Diminish cathepsin secretion Inhibit gelatinase activity Inhibit its dev’ment Induce Osteoclast apoptosis Decrease ruffled border Alter Intracellular Ca+2
CMT’S AND THEIR ACTIONS IN PERIODONTAL DISEASES • Gingival and Periodontal infection
BIOLOGICAL ROLES OF CMTS
IN INFLAMMATION
ACTIONS ON BONE
CMTS AS CHELATING AGENTS • Collagenase enzyme itself contains Zinc ions and it requires Calcium ions for it’s stabilization. • CMTs are ionophores or chelating agents that bind to Zn or Ca ions thereby inhibiting collagenase enzyme.
INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE (INOS) • CMTs may decrease the ROS burden by inhibiting neutrophils, directly scavenging free radicals and inhibiting reactions that lead to free radical generation. • They inhibit the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) activity.
• The peroxynitrite radical formed by the reaction of NO is highly cytotoxic, inhibits collagen and proteoglycan synthesis and up regulates the MMP expression. • Inhibition of iNOS production causes reduction in the peroxynitrite levels, thus preventing denatuartion of proteins. CMT-3 and CMT-8 have shown maximum inhibitory effect on the iNOS, CMT-1 and-2 had intermediary effect while CMT-5 was ineffective. • Trachtman .H.et.al(1998)
INHIBITION OF PROINFLAMMATORY MEDIATORS • CMTs inhibit release of IL-1β, IL-6, IL-8, TNF-α and PGE2 from LPS stimulated host immune cells by suppressing phosphorylation of the nuclear factor κ-B cell signalling pathway. The CMT-3 inhibits COX-2- mediated PGE-2 production. Golub .L.M et.al (1983)
• In an ex vivo human whole blood model stimulated with P. gingivalis LPS, doxycycline and CMT-3 were investigated for their efficacy in suppressing the production of proinflammatory mediators and MMPs. There was a significant reduction in the secretion of proinflammatory cytokines but the levels of MMPs were not affected. • Cazalis .J et.al (2009)
• Studies have shown that CMT -3 inhibited intracellular accumulation and synthesis of TNF-α in activated mast cells. It also inhibited IL-8 and protein kinase - C production. Protein kinase - C is an important mediator of transcription of MMPs, therefore inhibition of this mediator may produce an anti-inflammatory effect. Sandler .C .et.al.(2005)
98 Effect of CMT’s on LJP and Adult periodontitis patient’s: Drug conc. required for: Fibroblast interstitial collagenase is predominant type in GCF. LJP patient’s : 470µm in GCF of CMT-I, Adult periodontitis patient’s : 10 – 20 µm in GCF of CMT- I. because Neutrophil interstitial collagenase is predominant type in GCF. because
Action on P.gingivalis & T.denticola: Inhibits gingipain activities & Collagenolytic activity of P.gingivalis. Inhibited trypsin like activity of T.denticola. CMT-I inhibited serum albumin degradation by P.gingivalis & T.denticola. CMT-1 inhibited the inactivation of α1 proteinase inhibitor by P.gingivalis.
Greenwald et al. recently conducted a synergism study using CMT-1 + flurbiprofen, a standard nonsteroidal anti- inflammatory drug selected primarily because of its reported beneficial effect on bone loss in humans with adult periodontitis and the beagle dog model of periodontal disease. Synergistic Actions
OTHER USES OF CMT • Diabetes mellitus • Rheumatoid arthritis • Tumor metastasis • Other uses
DIABETES MELLITUS • Experiments in diabetic rats showed that daily oral administration of CMT for 21-37 days reduced levels of pathologically excessive collagenase in gingival tissues and skin. The CMTs also increased the skin collagen production as revealed by increased concentrations of hydroxyproline. • There was increased osteoblastic activity and bone formation. Golub LM, McNamara TF, D'Angelo G, Greenwald RA, Ramamurthy NS (1987)
• Both Type I and Type II diabetes mellitus have been related to periodontitis. In vitro and in vivo studies in rats with both the types of diabetes showed that the CMTs inhibited MMP activity, enzyme expression and alveolar bone loss. Administration of CMT-8 in type II diabetic rats with nephropathy or retinopathy showed a reduction in the incidence of cataract development, proteinuria, and tooth loss. • The results were better with CMTs as compared to the commercial tetracyclines. Ryan ME, Ramamurthy NS, Sorsa T, Golub LM.(1999)
RHEUMATOID ARTHRITIS • Rheumatoid arthritis is a chronic inflammatory disease primarily related to excessive collagenase and PGE2 production causing bone, joint or tissue destruction. • The PGE-2 increase local blood flow and potentiates the action of mediators such as bradykinin. • It affects the cellular functions causing activation of MMPs, induction of apoptosis, inhibition of chondrocytic growth, activation of osteoclastic bone resorption, upregulation of IL-1 transcription factor and cAMP levels.
• Both in vitro and in vivo studies have demonstrated the beneficial role of tetracyclines and CMT-1 in suppressing the collagenase activity in the cultured synovial tissue. However, a combination therapy using CMT-1 and flurbiprofen (nonsteroidal anti-inflammatory drug) produced a greater suppression of the clinical inflammation along with radiographic improvement of the joint condition. This was due to the synergistic effects of anti- inflammatory action of flurbiprofen along with anticollagenase action of CMT-1. • Greenwald et.al
• Besides CMT-1, CMT-3 and -8 have also shown inhibitory effect on COX-2-induced PGE2 production. Additional animal and human trials are however required to evaluate the efficacy of these agents in treatment of arthritis.
TUMOR METASTASIS • The CMTs kill tumor cells by generation of hydroxyl free radicals which permeate and depolarize mitochondria. They also activate caspase-mediated apoptosis and reduce the rate of angiogenesis. • Inhibition of the type IV collagenase prevents the tumor cells from invading the basement membrane barriers and hence metastasis. • Lokeshwar BL. (2014)
• In a phase II trial of CMT-3 in the treatment of Kaposi's sarcoma in HIV patients, a significant decrease in serum MMP-2 and MMP-9 levels was seen. It was suggested that high doses of CMT-3 (50-150 mgqd) may be beneficial in the Kaposis's sarcoma patients who did not respond to highly active antiretroviral therapy alone. • This may be attributed to its ability to inhibit neutrophil elastase. Dezube BJ, Krown SE, Lee JY, Bauer KS, Aboulafia DM(2006)
OTHER USES • CMT-3 has been shown to have antifungal properties. Liu Y . et.al (2002) • Lower oral doses of CMT-3 are effective in decreasing the severity of acne. Golub LM. (2011) • They have also been used in the treatment of life threatening conditions like epidermolysis bullosa and acute respiratory distress syndrome associated with excessive collagenase activity. (Steinberg J ,et al 2011)
CMT’s potential advantages over conventional Tetracyclines: The recent observations in rats shown that CMT-1 is absorbed after oral administration more rapidly and has a longer serum half life than tetracycline. Their long-term systemic administration does not result in gastrointestinal toxicity, No resistance. Can be used for prolonged periods.
Current status of CMT’s: • CMTs have not yet been approved for human use by the FDA, although the National Cancer Institute has recently initiated preliminary studies, using CMT-3, on humans with cancer. • More recent studies have demonstrated the therapeutic potential of TCs' anti-MMP activity in in vivo and cell culture models of cancer invasion, metastasis, and angiogenesis ( Masumori et al., 1994; Lokeshwar et al., 1997; Seftor et al., 1998).
CONCLUSION • Tetracycline and its analogues have been used in the treatment of various diseases. • Although there is some evidence for anti-inflammatory and immunomodulatory effects, additional studies must be performed, at both the laboratory and clinical levels, to corroborate these properties.
REFERENCES 1. Essentials of medical pharmacology,6th edition ,K.D Tripathi 2. Textbook of Microbiology and Immunology ;Subhash Chandra Parija 3. Carranza’s Clinical periodontology,11th edition 4. Ramamurthy NS, Golub LM, Gwinnett AJ, Salo T, Ding Y, Sorsa T. In vivo and in vitro inhibition of matrix metallopro- teinases including MMP-13 by several chemically modified tetracyclines (CMTs). In: Davidovitch Z, Mah J, eds. Biological Mechanisms of Tooth Eruption, Reabsorption and Replace- ment by Implants. Boston: Harvard Soc Adv Orthodont, 1998: 271/7. 5. Rajesh N. Patel, Mukundan G. Attur, Mandar N. Dave, Indravadan V. Patel, Steven A. Stuchin, Steven B. Abramson and Ashok R. Amin .A Novel Mechanism of Action of Chemically Modified Tetracyclines: Inhibition of COX-2-Mediated Prostaglandin E2 Production. The Journal of Immunology, 1999, 163: 3459-3467.
6. Lokeshwar BL, Seltzer MG, Dudak SM, Bloch LN, Golub LM. Inhibition of tumor growth and metastasis by oral administration of a non-antimicrobial tetracycline analog (CMT-3), and doxycycline in a metastatic prostate cancer model. Int J Cancer 2002; 98: 297/309. 7. Zeina Saikalia and Gurmit Singha. Doxycycline and other tetracyclines in the treatment of bone metastasis. Anti-Cancer Drugs 14:773–778 c 2003 Lippincott Williams & Wilkins. 8. Svein Steinsvoll . Periodontal Disease, Matrix Metalloproteinases and Chemically Modified Tetracyclines. Microbial Ecology in Health and Disease 2004; 16: 1/7
9. Golub LM, Lee HM, Nemiroff A, McNamara TF, Kaplan R, Ramamurthy NS. Minocycline reduces gingival collagenolytic activity during diabetes: preliminary observations and a proposed new mechanism of action. J Periodont Res 1983; 18: 516/26. 10.R.A. Greenwald. Stretching the Boundaries of Conventional Thought: Larry Golub and the Tetracycline Story. J dent res 1999 78: 820. 11.Allen N. Sapadin, MD, and Raul Fleischmajer, MD. Tetracyclines: Nonantibiotic properties and their clinical implications. J Am Acad Dermatol 2006;54:258-65.
THANK YOU

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Tetracycline and its modifications .pptx seminar 5

  • 1. TETRACYCLINE AND IT’S MODIFICATIONS PRESENTED BY : DR. MITALI . V. THAMKE I M.D.S
  • 2. INDEX 1. Introduction and background 2. Classification 3. Structural Activity Relationship 4. Mechanism of action of tetracyclines 5. Spectram activity 6. Uses of tetracyclines
  • 3. INDEX 7. SIDE EFFECT OF TETRACYCLINE 8. LOCAL DRUG DELIVERY 9. TETRACYCLINE AS A HOST MODULATING AGENT 10. CHEMICALLY MODIFIED TETRACYCLINE 11. CONCLUSION 12. REFERENCES
  • 4. Definition: Tetracyclines are octahydro napthacene derivatives which are bacteriostatic and broad spectrum antibiotics that kills certain infection - causing microorganisms and are used to treat wide variety of infections.
  • 5. The tetracyclines were first discovered in 1948 as fermentation product of golden colored soil bacterium named Streptomyces aureofacians. Chemical isolation and purification of its products produced the compound Chlortetracycline in 1948, the first tetracycline to be fully characterized both chemically and clinically. Its advantages were a larger spectrum of activity & was better-tolerated by & less toxic to some individuals. INTRODUCTION AND BACKGROUND:
  • 6. According to duration of action: Short-acting (Half-life is 6-8 hrs) • Tetracycline Chlortetracycline Oxytetracycline Intermediate-acting (Half-life is ~12 hrs) Long-acting (Half-life is 16 hrs or more) • Doxycycline Minocycline Tigecycline • Demeclocycline Methacycline
  • 7. 7
  • 8. 3rd generation (After 1972) 4th generation 1st generation (Until 1965) 2nd generation (1965-1972) Tetracycline; Chlortetecycline; Oxytetracycline; Demeclocycline; Glycylcycline Tigecycline Azatetracycline Alkylaminotetracyclin e Doxycycline; Lymecycline; Meclocycline; Methacycline; Minocycline; Rolitetracycline DEPENDING UPON THEIR SYNTHESIS
  • 9. OH OH CONH2 OH R1 O O R2 R3 H R4 H N(CH3)2 OH ABCD 1 2 3 4 4a 5 5a 6 6a 7 8 9 10 10a 11 11a 12 12a N(CH3)2 Increases activity Conversion to nitriles causes a 20 f old increase in activity Modif ication leads to loss of activity =CH2 Increases the Antibacterial activity Elimination of 6-OH group increase lipophilicity & more stable to acids. Ex: Doxycycline. ‘D’ ring should be always aromatic Changes in this ring Leads to biological inactivation of the molecule. Additional glycyl amino substitution at the 9th Position leads to the new Class of antibiotics the glycylcyclines. EX: Tigecycline.(Tygacil) The keto-enol tatomerism Between c2 and c3 are very important f or biological activity. Inviolate zone is essential The linearly f used tetracyclic nucleus is most important f or the antibiotic activity. Electron donating (or) electron withdrawing groups at c7 increased Antibacterial activity Substitution with –OH Produce water soluble derivatives which can be administered orally. Epimerization at c4 and dehydration at 5a results loss of activity. 9 Structural Activity Relationship:
  • 10.  Tetracyclines inhibit protein synthesis by binding to the bacterial ribosome involved in the translation(protein synthesis) process and making them bacteriostatic.  The bacterial ribosome is a 70s particle made up of 30s subunit and 50s subunit.  The 30s subunit binds mRNA and initiates the protein synthesis.  The 50s subunit combines with the 30s subunit-mRNA complex to form a ribisome then binds aminoacyl tRNA and catalyses the building of the protein chain..  There are two main binding sites for the tRNA molecule.  The peptidyl(p-site) binds the tRNA bearing the peptide chain  The acceptor aminoacyl site (A-site)  Tetracyclines reversibly bind to the 30S subunit at the A-site to prevent attachment of the amino acyl tRNA, terminating the translation process. . . 10
  • 11. 11
  • 12. ANIMATION ILLUSTRATING THE ROLE OF TETRACYCLINES IN BLOCKING TRANSLATION DURING BACTERIAL PROTEIN SYNTHESIS
  • 13. ABSORBTION DISTRIBUTION EXCRETION Absorbed from duodenum and small intestine. Forms insoluble complex with Ca+,Mg+,Al+,Zn+. Absorption of tetracycline is impaired by the concurrent use of dairy products. Mechanism responsible for decreased absorption appears to be chelation of divalent and trivalent cations. Throughout the body Cross the placenta &enter the fetal circulation &amniotic fluid Kidney through glomerular filteration(except minocycline)
  • 14. ROUTES OF ADMINISTRATION • Oral • Parentral • Topical
  • 15. THERAPEUTIC USES I. Antibacterial properties II. Non antibacterial properties
  • 16.  Gram +ve & -ve bacteria  Spirochetes  Mycoplasms  Rickettsiae  Candida Albicans  Mycoplasma Pneumoniae  Chlamydia Trachomatis  Borrelia Recurrentis  Yersinia Pestis  Vibrio Cholerae  Campylavacter Fetus  Brucella Specie  Streptococcus Pneumonia  Neisseerie Gonorrhoeae 16 • Tetracyclines are broad spectrum antibiotics. They are active against following micro organisms:
  • 17. NONANTIBACTERIAL PROPERTIES • Anti-inflammatory effect • Immunosuppressive property • Suppression of antibody production in lymphocyte • Reduction of phagocytic function of PMN • Reduction of leukocyte & neutrophil chemotaxis • Inhibition of lipase and collagenase activity • Enhancer of gingival fibroblast cell attachment • Antitumor activity
  • 18. ADVERSE EFFECTS 1. Teeth and bones : • Tetracycline chelates calcium, forming a tetracycline-orthophosphate complex and are deposited in area of calcification in bone and teeth • Administration in pregnant women • lead to yellow staining of teeth in infants; defective formation of enamel and hypoplasia of teeth may occur • Tetracycline deposit in foetal bones and may reduce linear growth • Also be avoided in infants and children upto age of 12 years
  • 19. • Tetracycline can cause skin reaction. • Tetracycline get deposited in nails,which may cause nails to fluroscence. • Photosensitivity manifested by marked erythema. • Superinfection : supression of the normal intestinal microflora is liable to occur after prolonged tetracycline therapy.
  • 20. • Mild nausea, vomiting, diarrhea. • White patches or sores inside your mouth or on your lips . • Swollen tongue, trouble swallowing. • Loss of appetite, • Jaundice (yellowing of the skin or eyes). 20
  • 21. Drug Possible sequence Penicillin Antagonism of bactericidal action Antacids &iron preparation Impaired absorption of TCl insulin Oxytetracycline enhances hypoglycemic action Contraceptive pills Pill failure Cimetidine Reduced bioavailablity of TCl Digoxin  in serum digoxin concentration Lithium carbonate  serum level of lithium Warfarin sodium Enhanced anticoagulant effect Carbemazapine and phenytoin Reduced serum concentration of Doxycline DRUG INTERACTIONS
  • 22. RESISTANCE TO TETRACYCLINE 3 MAIN RESISTANCE MECHANISM: • accumulation of tetracycline a)antibiotic influx b)energy dependent efflux pathway • Presence of ribosomal protection proteins • Enzymatic inactivation of tetracycline (Speer et al 1992)
  • 23. • tet gene responsible for tetracycline resistance • tet (B)-coding for efflux • tet(M)-ribosomal protection protein • tet(X)-Enzymatic alteration
  • 24. SYSTEMIC TCL THERAPY IN PERIODONTAL USE
  • 25. CHRONIC & AGGRESSIVE PERIODONTITIS IN THE ADULT PATIENTS • Systemic Tetracycline –for 30 days produce  in clinical attachment level& in probing depths (Haffajee et al 1995) • Disadvantages • No statistically significant difference were measured in sub gingival composition between DOXY &placebo patient. • Recurrent disease activity
  • 26. REFRACTORY PERIODONTITIS • Drug regimen • Doxycline for 3 week • Reduced relative risk for periodontal breakdown over a seven month period by 43% (Mc cullon et al 1990)
  • 27. DISADVANTAGES • Recurrent disease activity (Van winkelhoft 1989) • Subgingival microorganism not sufficiently suppresed (Muculloh 1990) • No difference in subgingival microflora between placebo and DOXY treated (Kukarni et al 1991) •  in the number of organism (Muller 1990) • Super infection and opportunistic pathogens (Brogd et al 1985)
  • 28. AGGRESSIVE LOCALISED PERIODONTITIS • Drug regimen • Tetracycline HCl(250mg four times daily for 14 days every 8weeks) or DOXY 100mg 2 tablets stat and then single tablet for14 days. • Periodontal surgery together with systemic Tcl-Hcl were able to decrease the periodontal AA below detectable level. (Slots &Rosling 1988) • ADVANTAGES • Reduced gingival inflammation • Gain in clinical attachment and alveolar bone (Genco et al )
  • 29. DISADVANTAGES • Reactivation of disease (25%) (Lindhe 1982) • Periodontal destruction was noted with post treatment levels (Slots & Rosling 1993) • TCl did not suppress AA in all LJP
  • 30. MOUTH RINSE SUBGINGIVAL IRRIGATION SYSTEMIC DELIEVERY CONTROLLED DELIVERY Reaches site of disease activity Poor Good Good Good Adequate drug concentration Good Good Fair Good Adequate duration of therapy Poor Poor Fair Good DIFFERENT DRUG DELIVERY SYSTEM
  • 32. KORNMAN2 CLASSIFIED CONTROLLED RELEASE DELIVERY SYSTEMS IN PERIODONTICS BASED ON THE RELEASE OF THE DRUG INTO:RESERVOIR WITHOUT RATE CONTROLLING SYSTEM: RESERVOIR DEVICES WITH RATE CONTROLLING SYSTEM: Includes devices such as hollow fibers filled with a therapeutic agent in which the agent is released simply by diffusion through the reservoir wall. The most common forms include solvent action on coated drug particles, microporous polymer membranes or monolithic matrices, or erodable polymeric matrices. Classification based on the rate controlling system Kornman
  • 33. 2.RAMS & SLOTS (1996): DEPENDING ON THE USAGE PERSONALLY APPLIED (In patient home self-care) PROFESSIONALLY APPLIED(In dental office)
  • 34. BASED ON DURATION OF ACTION (GREENSTEIN & TONETTI 2000) A) SUSTAINED RELEASE DEVICES Drug delivery for less than 24 hrs require multiple applications follow first order kinetics B) CONTROLLED DELIVERY DEVICES Duration of drug release exceeds 24hrs administered once follow zero order kinetics
  • 35. INDICATIONS: CONTRA-INDICATIONS:  Periodontal patients with known hypersensitivity reaction to any of the antimicrobials used for periodontal therapy.  Patients susceptible to infective endocarditis are contraindicated for irrigation devices to avoid the risk of bacteremia.  Delivery of antimicrobial agents using ultrasonic scalers is contraindicated in asthmatics, infective conditions (AIDS, TB) and those with cardiac pacemakers  Isolated periodontal pockets (>5mm), with successful phase 1 therapy  Periodontal patients who are medically compromised where surgical therapy is contraindicative.  In combination with mechanical debridement or alone.  In-patients who are suffering from recurrent or refractory periodontitis.  During periodontal regenerative procedures.
  • 36. • Advantanges: 1. Attains a 100- fold higher concentration of anti micro- bial agent in subgingival sites. 2. Reduces patient dose by over 400 fold thereby reduc- ing chances of drug resistance and side effects caused by systemic antibiotics 3. Small doses can be administered. 4. Maintain contact with the pathogens in the infected area for a prolonged period of time. 5. Maintains effective concentration. Goodson J (2000)
  • 37. • Disadvantages: 1. Patient may not comply to placement of the drug sub- gingivally. 2. Diffculty in placing the device at the base of the pocket. 3. Lack of manual dexterity. 4. Does not have any effect on adjacent or nearby structures such as tonsils, buccal mucosa etc so may cause chances of re- infection. 5. Time consuming 6. Costly
  • 38. PROBLEMS • Flushing action of GCF • Small volume of periodontal pocket • Pressure exerted by tonus of periodontal tissue • Drug reservoir in the periodontal pocket will be depleted within hours or days after placement
  • 39. LOCAL DELIVERY DEVICES Tetracycline are available in following local delivery devices : 1. Fibers 2. Films 3. Gels 4. Strip 5. Microparticles 39
  • 40. COMMERCIALLY AVAILABLE PRODUCTS 1. Tetracycline fibers (Actisite, Alza Corp., Mountain view, California) 2. Minocycline ointment (Dentomycine, Lederle, UK & Periocline, Sunstar, Japan) 4. Doxycycline hyclate in a resorbable polymer (Atridox, Atrix Labs, CO) 6. Minocycline microspheres (Arestin, Ora pharma, North Carolina, USA).
  • 41. TETRACYCLINE –CONTAINING FIBERS(ACTISITE) • FDA approved • Non resorbable, biologically inert, safe plastic copolymer (ethylene &vinyl acetate) loaded with 25% w/w tetracycline hcl powder packed as thread of 0.5mm diameter &23cm length • It maintains constant concentrations of active drug in the crevicular fluid in excess of 1000 μg/mL for a period of 10 days.(Maurizio S etal) • In contrast GCF conc. of only 4-8 microgram/ml were reported after systemic administration, 250 mg qid for 10 days.
  • 42. • Bioresorbable form is PERIODONTAL PLUS AB • Biodegrade within 7 days ,so no 2nd appointment • Gel (Maheshwari etal) • Tetracycline-Serratiopeptidase-Containing Periodontal Gel • Formulation has shown statistically significant results along with scaling and root planing.
  • 43. • STUDIES • Singh et al.(2009)- 3 months - No difference in the results achieved with local tetracycline hydrochloride or local metronidazole as adjuncts to mechanotherapy. • However, both antibiotic therapies resulted in greater improvement in microbiological parameters when compared to mechano therapy alone.
  • 44. • Sadaf et al.(2012)-Tetracycline fibers- 3 months • Higher reduction in plaque index, gingival index and in the clinical probing depths of the tested group than of the control group at all time intervals -15, 30, 60 and 90 days. • Tetracycline fiber therapy enhances the benefits of SRP in the treatment of chronic periodontitis. (Gupta Nidhi et al ,2015)
  • 45. SUBGINGIVAL DOXYCYCLINE • Doxycycline is a bacteriostatic agent • Has the ability to down regulate MMP’s . • The only FDA approved 10% Doxycycline in a gel system ATRIDOX (42.5 mg Doxycycline) is a subgingival controlled-release product composed of a 2 syringe mixing system. It is the only local delivery system accepted by ADA
  • 46. • Doxycycline levels in GCF peaked to 1,500 - 2000 μg/ml in 2 hours following treatment with ATRIDOX. • Local levels of Doxycycline have been found to remain well above the MIC for periodontal pathogens (6.0μg/ml) through Day 7. • 95% of the polymer is bio absorbed or expelled from the pocket naturally within 28 days.
  • 47. • Locally applied controlled release DOX gel may partly counteract the negative effect of smoking on periodontal healing following no surgical therapy.(Tomasi C and Jan LW)
  • 48. STUDIES  The FDA has also approved doxycycline hyclate in a bioabsorbable polymer gel as a stand-alone therapy for the reduction of probing depths, bleeding upon probing, and gain of clinical attachment (Kim TS etal,2002)  Deo et al.(2011)- 6 months - Doxycycline hyclate 10% as an adjunct to SRP provided significant reductions in PPD and gains in CAL compared to SRP alone.
  • 49. SUBGINGIVAL MINOCYCLINE • A bacteriostatic antibiotic has been tried clinically via in three different modes i.e. film, microspheres, and ointment. Film: • Ethyl cellulose film containing 30% of Minocycline were tested as sustained release • complete eradication of pathogenic flora from the pocket after 14 days
  • 50. Microsphere: • A new, locally delivered, sustained release form of minocycline microspheres (ARESTIN) for subgingival placement is available. • The 2% minocycline is encapsulated into bio-resorbable microspheres (20-60μm in diameter) in a gel carrier and has resorption time of 21 days. • Gingival crevicular fluid hydrolyses the polymer and releases minocycline for a period of 14 days or longer before resorbing completely.
  • 51. Ointment: • 2% minocycline hydrochloride in a matrix of hydroxyethyl-cellulose, amino alkyl-methacrylate, triacetine & glycerine. • DENTOMYCIN –european union • PERIOCLINE -JAPAN • The concentration of minocycline in the periodontal pocket is about 1300μg/ml, 1 hr after single topical application of 0.05 ml ointment (1mg of minocycline) and is reduced to 90μg/ml after 7 hrs.
  • 52. STUDIES • Timmerman et al (1996) reported that there was no benefit of employing 2% minocycline gel as an adjunct to SRP to reduce probing depths at deep sites • Steenberghe et al (1999) that combined therapy provided a better result than SRP alone at sites >7 mm deep. • Jung et al.( 2012) -Minocycline hydrochloride2% -Reductions in PPD, BOP and gain in CAL were significantly greater at the minocycline ointment in association with flap surgery site than at the flap surgery site alone
  • 53. ISSUE SYSTEMIC ADMINISTRATION LOCAL ADMINISTRATION Drug distribution Wide distribution Narrow effective range Drug concentration Variable levels High dose at treated site,low levels elsewhere Therapeutic potential Reach widely Act better locally Problems Side effects Reinfection from non- treated site Clinical limitations Require good patient compliance Infection limited to treated site Diagnostic problem Identification of pathogens Identification of sites to be treated COMPARISON OF SYSTEMIC &LOCAL DRUG DELIVERY
  • 54. HOST MODULATING THERAPEUTIC AGENTS • Sub Antimicrobial Dose of Doxycycline • Chemically modified Tetracyclines(CMT). • Synthetic –antiMMPs • Bisphosphonates. • NSAIDs-
  • 55. LOW DOSE TETRACYCLINES • Golub et al recommended - 20 mgs Doxy capsules,twice a day for 2 weeks . • This low dose had no antimicrobial effect and hence did not lead to development of resistant microorganisms in sub gingival plaque.[Golub 1985,Thomas 1998,Walker et al 2000]. • However the anticollagenase activity was unaffected &significantly reduced collagenase activity in GCF was demontrated.[Golub 1990]. • Croat et al 1996 showed improved clinical parameters [attachment level , probing depth] when administered periodically over 6 month period.
  • 56. SUB ANTIMICROBIAL DOSE OF DOXYCYCLINE • Doxycycline hyclate (Periostat): "Research is in My Blood" Dr. Lorne Golub, Developer of Periostat, received the ADA's Gold Medal Award for Excellence in Dental Research in 2006. • Available as 20-mg capsule, prescribed twice daily for use. • Approved by US Food and Drug Administrator (FDA), for the adjunctive treatment of periodontitis. • It acts by suppression of the activity of collagenase, particularly that produced by polymorphonuclear neutrophils. • It does not exhibit antimicrobial effects but can effectively lower MMP level
  • 57. • Mcnamara et al attributed the lack of side effects to the 92% reduction in blood levels of the drug in patients on the low- dose doxycycline • Goldberg et al. found that Combination of SDD plus low-dose flurbiprofen, together with repeated bouts of nonsurgical periodontal therapy, produces improvements in nonresponsive patients (refractory periodontitis).
  • 58. LIMITATION OF SDD • Either composition or resistant levels of microflora was unaltered. (walker et al 2000) • More aggressive forms of collagenolytic disease can’t be controlled.
  • 59. ANTI-COLLAGENESE EFFECT • Inhibit active collagenase from the host • Prevention of conversion of pro-MMP to active MMP • Down regulation of pro-MMP expression • Excessive proteolysis of pro-MMP into enzymatically inactive fragments. • Prevent MMP’s from degrading/inactivating the serum protein 1 – proteinase inhibitor. (Golub et al 1990, 1994) • Collagenase produced from PMN (MMP-8)than from fibroblast (MMP-1).
  • 61. The unexpected ability of tetracyclines to inhibit the breakdown of connective tissue & bone by a non-antimicrobial mechanism was first reported over 6 decades ago by McCormick 1957, Wilkinson, 1958, & more recently, produced & characterized by the group of McNamara, T. F., Golub, L. M., D'Angelo, G.,& Ramamurthy, 1986. Based on the thoroughly explored chemistry of the tetracyclines, a number of tetracycline analogs can be synthesized with side-chain deletions or, in some cases, moieties added to the parent tetracycline molecule.
  • 62. Golub and co-workers 1983,84 made the seminal observation that TC’s inhibit collagenase activity and, thereby, launched the current interest in their non-antimicrobial properties. Their studies showed that: 1) Severely hyperglycemic, diabetic rats had reproducible, elevated levels of collagenase activity in gingiva (and skin); and 2) This increase in enzyme activity probably led to severe periodontal breakdown.
  • 63. 3) Furthermore, such an increase likely resulted, at least in part, from a shift to Gram-negative microflora in the subgingival plaque with more of its bacterial endotoxin concentrated in the gingival sulcus. Endotoxin penetration into the subepithelial connective tissue could then stimulate host cells; i.e., fibroblasts, macrophages, and keratinocytes to produce collagenase.
  • 64. 67 Golub and co-workers modified their experimental diabetes protocol using tetracycline therapy and germ-free rats to determine whether the Gram-negative microflora increased collagenase levels. The Diabetic Rat Model:
  • 65. 68 Conventional Rats Gnotobiotic Rats Periodontitis & Diabetes Minocycline 65% of pathologically excessive mammalian collagenase activity was reduced. Diabetes Minocycline 70% of diabetic gingival collagenase activity was reduced.
  • 66. As a result of these initial studies, Golub et al. proposed: (1)That tetracyclines, but not other antibiotics, can inhibit collagenase by a mechanism not dependent on the drug's antibacterial efficacy & (2) That this newly discovered property of tetracyclines could provide a new approach to the treatment of diseases, such as periodontal disease, & also including other (medical) disorders like rheumatoid and osteoarthritis (as well as several cutaneous and other diseases) that involve collagen destruction.
  • 67. 70 Tetracyclines are known to inhibit collagenases (& some other, but not all, matrix metalloproteinases or MMPs from a variety of cells: Neutrophils, Macrophages, Osteoblasts, Chondrocytes, and A wide range of tissues: skin, gingiva, cornea, cartilage, and rheumatoid synovium.
  • 68. Tetracyclines inhibit PMN but not fibroblas collagenase: PMN’s Collagenase 2-25µg/ml of Minocycline Inhibited Fibroblast Collagenase 250µg/ml Minocycline required Not Inhibited
  • 69. Tetracyclines inhibit PMN but not fibroblast collagenase • It has been suggested that PMN’s provide the major source of collagenase that mediates tissue breakdown during inflammatory periodontal disease, • Fibroblasts contribute the collagenase required for connective tissue remodeling in normal gingiva. • Therapy with these drugs would be expected to reduce pathologically elevated collagenolytic activity (e.g., during inflammation), but not the collagen turnover required to maintain normal tissue integrity.
  • 70. 73 To identify the site of the anticollagenase property, Golub and co-workers 1991, synthesized 10 different analogs of tetracyclines known as chemically-modified tetracyclines (CMTs 1-10). All 10 analogs lacked antimicrobial efficacy and inhibited collagenase activity, but only 1 did not.
  • 71. STRUCTURAL FORMULAE OF THE TETRACYCLINE OH O OH O O C NH2 OH N(CH3)2 HHOHCH3 OH 10 9 8 7 6 11 12 5 1 2 3 4 Site of antimicrobial action. Golub et al removed the dimethyl amino group from the C-4 atom and this Chemically modified tetracycline.[CMT].lost all its anti microbial property but retained its anticollagenase effect.
  • 72. O OH OHCH3 OH OH O OH CONH2 CMT-1 [4-Dedimethylamino tetracycline] CMT-7 [12a-Deoxy-4-dedimethylamino tetracycline] O OH OHCH3 OH O OH CONH2 4 3 2 1 12 a
  • 73. 76 CMT-2 or tetracyclinonitrile was produced by dehydration of the carboxamide residue at carbon 2. CMT-3 - produced by removing the hydroxyl & methyl groups on carbon 6 & 4.
  • 74. 77 CMT- 4 - produced by removal of dimethylamino group from carbon 4 of chlortetracycline. when the carbonyl oxygen at carbon 11 and hydroxyl group at carbon 12 were removed by converting tetracycline to the pyrazole derivative or CMT-5, as described by Valcavi et al., the collagenase- inhibitory activity of the molecule was lost.
  • 75. CMT-5-TETRACYCLINE PYRAZOLE O OH N(CH3)2 OHCH3 OH OH N N CONH2 CMT-5 (the pyrazole analogue of TC) (Valcavi et al,1963), which lacks the Ca2+ and Zn2+ binding site at carbon-11 and carbon-12 of the TC molecule,does not inhibit MMP activity, but it retains its ability to prevent pro-collagenase activity by HOCL inhibition. ( ROS-reactive oxygen species) - Sorsa et al 1998
  • 76. CMT-8 ,the non-antimicrobial analog of doxycycline, appears to increase the MMP-inhibitory efficacy ,matrix sparing activities. (Paemen et al,1996; Zernicke et al,1997; Greenwald et al, 1998; Ohyori et al 1998) CMT8>CMT3>CMT1 O OH OHCH3 OH OH OH CONH2 O CMT-8 [6a-deoxy,5hydroxy,4-Dedimethylamino doxycycline]
  • 77. 80 Mechanism of action of CMTs: The proposed mechanism of action of CMT’s results from their ability to bind metal ions, particularly Ca2+ and Zn2+, which are required by enzymes to maintain its proper conformation and hydrolytic activity. Inhibition of active or pro-MMP could occur due to chelation of Zn2+ ions at the binding site in the catalytic domain, resulting in disruption of normal conformation of protein structure, & leaving it non-functional due to excessive degradation into small molecular weight, enzymatically inactive fragments.
  • 78. 81 CMTs have also been shown to downregulate expression of gelatinases, & thus to reduce the production of pro- enzyme (MMP-2 and MMP-9) (Golub et al., 1991, 1998). Also, CMTs may inhibit the activation of collagenases (MMP-1, MMP-8, and MMP-13), and Inhibit Stromelysins (MMP-3, MMP-10,and MMP-11) and Inhibit MT-MMP’s,
  • 81. PMN’s Osteoblasts Procollagenase Collagenase ROS e.g. HOCL by neutrophils Secretes Activated CMT-1 100-400 µm Inhibits 40 – 80% TC effects on pro- collagenase activation:
  • 82. Other mechanisms that have been proposed include: Inhibition of oxidative activation and increase in degradation of pro- MMP's, Inhibition of production of secretary non-pancreatic phospholipase A2, Inhibition of cytokine production, i.e., of TNF-alpha and IL-8, and Reduction of the expression of serine proteinase and trypsinogen-2. (Pruzanski et al., 1998; Kirkwood et al., 1999). Inhibition of protein glycation, and Inhibition of non-collagenolytic proteases. Inhibit secretion of other collagenolytic enzymes like Lysosomal cathepsin.
  • 83. 86 Bone resorption Inhibition: As examples of in vitro efficacy, TCs and CMTs were found to inhibit bone resorption in both organ and cell culture, regardless of whether the resorption was induced by parathyroid hormone (PTH), PGE2, or bacterial endotoxin (Golub et al, 1984; Gomes et al, 1984; Rifkin et al, 1994). CMT-1 and -3 and CMTs-6, -7, and -8 were effective inhibitors of bone resorption in culture (CMT-8 was the single most potent compound), whereas CMT-2, -4, and -5 were not.
  • 84. 87 Tc’s affect several parameters of Osteoclast function: Effects on Osteoclast function Diminish acid production Diminish cathepsin secretion Inhibit gelatinase activity Inhibit its dev’ment Induce Osteoclast apoptosis Decrease ruffled border Alter Intracellular Ca+2
  • 85. CMT’S AND THEIR ACTIONS IN PERIODONTAL DISEASES • Gingival and Periodontal infection
  • 89. CMTS AS CHELATING AGENTS • Collagenase enzyme itself contains Zinc ions and it requires Calcium ions for it’s stabilization. • CMTs are ionophores or chelating agents that bind to Zn or Ca ions thereby inhibiting collagenase enzyme.
  • 90. INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE (INOS) • CMTs may decrease the ROS burden by inhibiting neutrophils, directly scavenging free radicals and inhibiting reactions that lead to free radical generation. • They inhibit the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) activity.
  • 91. • The peroxynitrite radical formed by the reaction of NO is highly cytotoxic, inhibits collagen and proteoglycan synthesis and up regulates the MMP expression. • Inhibition of iNOS production causes reduction in the peroxynitrite levels, thus preventing denatuartion of proteins. CMT-3 and CMT-8 have shown maximum inhibitory effect on the iNOS, CMT-1 and-2 had intermediary effect while CMT-5 was ineffective. • Trachtman .H.et.al(1998)
  • 92. INHIBITION OF PROINFLAMMATORY MEDIATORS • CMTs inhibit release of IL-1β, IL-6, IL-8, TNF-α and PGE2 from LPS stimulated host immune cells by suppressing phosphorylation of the nuclear factor κ-B cell signalling pathway. The CMT-3 inhibits COX-2- mediated PGE-2 production. Golub .L.M et.al (1983)
  • 93. • In an ex vivo human whole blood model stimulated with P. gingivalis LPS, doxycycline and CMT-3 were investigated for their efficacy in suppressing the production of proinflammatory mediators and MMPs. There was a significant reduction in the secretion of proinflammatory cytokines but the levels of MMPs were not affected. • Cazalis .J et.al (2009)
  • 94. • Studies have shown that CMT -3 inhibited intracellular accumulation and synthesis of TNF-α in activated mast cells. It also inhibited IL-8 and protein kinase - C production. Protein kinase - C is an important mediator of transcription of MMPs, therefore inhibition of this mediator may produce an anti-inflammatory effect. Sandler .C .et.al.(2005)
  • 95. 98 Effect of CMT’s on LJP and Adult periodontitis patient’s: Drug conc. required for: Fibroblast interstitial collagenase is predominant type in GCF. LJP patient’s : 470µm in GCF of CMT-I, Adult periodontitis patient’s : 10 – 20 µm in GCF of CMT- I. because Neutrophil interstitial collagenase is predominant type in GCF. because
  • 96. Action on P.gingivalis & T.denticola: Inhibits gingipain activities & Collagenolytic activity of P.gingivalis. Inhibited trypsin like activity of T.denticola. CMT-I inhibited serum albumin degradation by P.gingivalis & T.denticola. CMT-1 inhibited the inactivation of α1 proteinase inhibitor by P.gingivalis.
  • 97. Greenwald et al. recently conducted a synergism study using CMT-1 + flurbiprofen, a standard nonsteroidal anti- inflammatory drug selected primarily because of its reported beneficial effect on bone loss in humans with adult periodontitis and the beagle dog model of periodontal disease. Synergistic Actions
  • 98. OTHER USES OF CMT • Diabetes mellitus • Rheumatoid arthritis • Tumor metastasis • Other uses
  • 99. DIABETES MELLITUS • Experiments in diabetic rats showed that daily oral administration of CMT for 21-37 days reduced levels of pathologically excessive collagenase in gingival tissues and skin. The CMTs also increased the skin collagen production as revealed by increased concentrations of hydroxyproline. • There was increased osteoblastic activity and bone formation. Golub LM, McNamara TF, D'Angelo G, Greenwald RA, Ramamurthy NS (1987)
  • 100. • Both Type I and Type II diabetes mellitus have been related to periodontitis. In vitro and in vivo studies in rats with both the types of diabetes showed that the CMTs inhibited MMP activity, enzyme expression and alveolar bone loss. Administration of CMT-8 in type II diabetic rats with nephropathy or retinopathy showed a reduction in the incidence of cataract development, proteinuria, and tooth loss. • The results were better with CMTs as compared to the commercial tetracyclines. Ryan ME, Ramamurthy NS, Sorsa T, Golub LM.(1999)
  • 101. RHEUMATOID ARTHRITIS • Rheumatoid arthritis is a chronic inflammatory disease primarily related to excessive collagenase and PGE2 production causing bone, joint or tissue destruction. • The PGE-2 increase local blood flow and potentiates the action of mediators such as bradykinin. • It affects the cellular functions causing activation of MMPs, induction of apoptosis, inhibition of chondrocytic growth, activation of osteoclastic bone resorption, upregulation of IL-1 transcription factor and cAMP levels.
  • 102. • Both in vitro and in vivo studies have demonstrated the beneficial role of tetracyclines and CMT-1 in suppressing the collagenase activity in the cultured synovial tissue. However, a combination therapy using CMT-1 and flurbiprofen (nonsteroidal anti-inflammatory drug) produced a greater suppression of the clinical inflammation along with radiographic improvement of the joint condition. This was due to the synergistic effects of anti- inflammatory action of flurbiprofen along with anticollagenase action of CMT-1. • Greenwald et.al
  • 103. • Besides CMT-1, CMT-3 and -8 have also shown inhibitory effect on COX-2-induced PGE2 production. Additional animal and human trials are however required to evaluate the efficacy of these agents in treatment of arthritis.
  • 104. TUMOR METASTASIS • The CMTs kill tumor cells by generation of hydroxyl free radicals which permeate and depolarize mitochondria. They also activate caspase-mediated apoptosis and reduce the rate of angiogenesis. • Inhibition of the type IV collagenase prevents the tumor cells from invading the basement membrane barriers and hence metastasis. • Lokeshwar BL. (2014)
  • 105. • In a phase II trial of CMT-3 in the treatment of Kaposi's sarcoma in HIV patients, a significant decrease in serum MMP-2 and MMP-9 levels was seen. It was suggested that high doses of CMT-3 (50-150 mgqd) may be beneficial in the Kaposis's sarcoma patients who did not respond to highly active antiretroviral therapy alone. • This may be attributed to its ability to inhibit neutrophil elastase. Dezube BJ, Krown SE, Lee JY, Bauer KS, Aboulafia DM(2006)
  • 106. OTHER USES • CMT-3 has been shown to have antifungal properties. Liu Y . et.al (2002) • Lower oral doses of CMT-3 are effective in decreasing the severity of acne. Golub LM. (2011) • They have also been used in the treatment of life threatening conditions like epidermolysis bullosa and acute respiratory distress syndrome associated with excessive collagenase activity. (Steinberg J ,et al 2011)
  • 107. CMT’s potential advantages over conventional Tetracyclines: The recent observations in rats shown that CMT-1 is absorbed after oral administration more rapidly and has a longer serum half life than tetracycline. Their long-term systemic administration does not result in gastrointestinal toxicity, No resistance. Can be used for prolonged periods.
  • 108. Current status of CMT’s: • CMTs have not yet been approved for human use by the FDA, although the National Cancer Institute has recently initiated preliminary studies, using CMT-3, on humans with cancer. • More recent studies have demonstrated the therapeutic potential of TCs' anti-MMP activity in in vivo and cell culture models of cancer invasion, metastasis, and angiogenesis ( Masumori et al., 1994; Lokeshwar et al., 1997; Seftor et al., 1998).
  • 109. CONCLUSION • Tetracycline and its analogues have been used in the treatment of various diseases. • Although there is some evidence for anti-inflammatory and immunomodulatory effects, additional studies must be performed, at both the laboratory and clinical levels, to corroborate these properties.
  • 110. REFERENCES 1. Essentials of medical pharmacology,6th edition ,K.D Tripathi 2. Textbook of Microbiology and Immunology ;Subhash Chandra Parija 3. Carranza’s Clinical periodontology,11th edition 4. Ramamurthy NS, Golub LM, Gwinnett AJ, Salo T, Ding Y, Sorsa T. In vivo and in vitro inhibition of matrix metallopro- teinases including MMP-13 by several chemically modified tetracyclines (CMTs). In: Davidovitch Z, Mah J, eds. Biological Mechanisms of Tooth Eruption, Reabsorption and Replace- ment by Implants. Boston: Harvard Soc Adv Orthodont, 1998: 271/7. 5. Rajesh N. Patel, Mukundan G. Attur, Mandar N. Dave, Indravadan V. Patel, Steven A. Stuchin, Steven B. Abramson and Ashok R. Amin .A Novel Mechanism of Action of Chemically Modified Tetracyclines: Inhibition of COX-2-Mediated Prostaglandin E2 Production. The Journal of Immunology, 1999, 163: 3459-3467.
  • 111. 6. Lokeshwar BL, Seltzer MG, Dudak SM, Bloch LN, Golub LM. Inhibition of tumor growth and metastasis by oral administration of a non-antimicrobial tetracycline analog (CMT-3), and doxycycline in a metastatic prostate cancer model. Int J Cancer 2002; 98: 297/309. 7. Zeina Saikalia and Gurmit Singha. Doxycycline and other tetracyclines in the treatment of bone metastasis. Anti-Cancer Drugs 14:773–778 c 2003 Lippincott Williams & Wilkins. 8. Svein Steinsvoll . Periodontal Disease, Matrix Metalloproteinases and Chemically Modified Tetracyclines. Microbial Ecology in Health and Disease 2004; 16: 1/7
  • 112. 9. Golub LM, Lee HM, Nemiroff A, McNamara TF, Kaplan R, Ramamurthy NS. Minocycline reduces gingival collagenolytic activity during diabetes: preliminary observations and a proposed new mechanism of action. J Periodont Res 1983; 18: 516/26. 10.R.A. Greenwald. Stretching the Boundaries of Conventional Thought: Larry Golub and the Tetracycline Story. J dent res 1999 78: 820. 11.Allen N. Sapadin, MD, and Raul Fleischmajer, MD. Tetracyclines: Nonantibiotic properties and their clinical implications. J Am Acad Dermatol 2006;54:258-65.