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Zašto tetraciklin ima tako širok spektar djelovanja?

Zašto tetraciklin ima tako širok spektar djelovanja?



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Koje su strukturne i kemijske karakteristike koje čine tetraciklin jedinstveno širokim spektrom? Razumijem da djeluje na A-mjesto prokariotskog ribosoma, ali postoje mnogi lijekovi usmjereni na ribosome koji su relativno uskog spektra. Pretpostavljam da ima neke veze s njegovom propusnošću u nekoliko različitih klasa bakterijskih staničnih stijenki i/ili membrana - ali ne mogu pronaći literaturu koja povezuje neki karakterističan dio njezine strukture s tom propusnošću.


Ovo je zeznuto pitanje. Prije svega, ne bih tetracikline nazvao "jedinstvenim" širokim spektrom. Oni su širokog spektra, ali postoje i drugi lijekovi s jednako širokom pokrivenošću bez stečene rezistencije (npr. imipenem i kloramfenikol), a trenutna klinička upotreba tetraciklina donekle je ograničena širenjem rezistencije. Ipak, nemaju nikakva veća klasna ograničenja. Opet, bez stečene rezistencije, tetraciklini su učinkoviti protiv gram pozitivnih aeroba i anaeroba, te gram negativnih organizama, kao što vam je poznato.

Tetracilini pristupaju svojoj meti na 30S podjedinici bakterijskog ribosoma (blokirajući mjesto A, kao što ste rekli) pasivnom difuzijom kroz porine vanjske membrane gram negativnih bakterija i aktivnim transportom kroz citoplazmatsku membranu gram negativnih i gram pozitivnih bakterija , ali mehanizam tog aktivnog transporta još uvijek je nepoznat. Ovo je prilično dobra recenzija iako je prilično stara. Sada, nekih 30 godina kasnije, još uvijek nismo identificirali točan mehanizam, kao što možete pročitati u najnovijem izdanju Goodmana & Gillmana (13.), Ch. 59, prelazak citoplazmatske membrane zahtijeva metaboličku energiju, ali proces nije dobro shvaćen. Zbog toga ne možemo ukazati na određenu strukturnu ili kemijsku karakteristiku. Za kloramfenikol, to bi bila sposobnost difuzije kroz citoplazmatsku membranu; za tetraciklin, ne znamo. Pretpostavljam da je dio razloga za to taj što su ljudi sada više zainteresirani za pumpe koje uklanjaju tetracikline iz stanice nego one koje ih akumuliraju, pa je to više pitanje o prioritetima istraživanja nego o nekom transportnom mehanizmu koji je posebno teško otkriti.


Tetraciklin

Tetraciklin, prodaje se pod robnom markom Sumycin među ostalim, oralni je antibiotik iz tetraciklinske obitelji lijekova, koji se koristi za liječenje brojnih infekcija, [1] uključujući akne, koleru, brucelozu, kugu, malariju i sifilis. [1]

    60-54-8 Y
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Uobičajene nuspojave uključuju povraćanje, proljev, osip i gubitak apetita. [1] Ostale nuspojave uključuju loš razvoj zuba ako ga koriste djeca mlađa od osam godina, probleme s bubrezima i lako izgaranje od sunca. [1] Upotreba tijekom trudnoće može naštetiti bebi. [1] Djeluje tako što inhibira sintezu proteina u bakterijama. [1]

Tetraciklin je patentiran 1953. i ušao u komercijalnu upotrebu 1978. [2] Nalazi se na popisu esencijalnih lijekova Svjetske zdravstvene organizacije. [3] Tetraciklin je dostupan kao generički lijek. [1] Tetraciklin je izvorno napravljen od bakterija Streptomyces tip. [1]


TETRACIKLINSKI ANTIBIOTICI Puni tekst

Tetraciklinski antibiotici imaju širok spektar djelovanja, relativno su sigurni, mogu se koristiti na mnogo načina primjene i široko se koriste. Imaju čak i antiprotozoalnu aktivnost. Glavna razlika između tetraciklina je u njihovim farmakokinetičkim svojstvima. Unakrsni otpor među članovima grupe je čest.

Struktura i kemijske karakteristike

Četiri spojena 6-eročlana prstena, kao što je prikazano na popratnoj slici, čine osnovnu strukturu od koje su napravljeni različiti tetraciklini. Različiti derivati ​​su različiti na jednom ili više od četiri mjesta na krutoj, planarnoj prstenastoj strukturi. Klasični tetraciklini su izvedeni iz Streptomyces spp., ali noviji derivati ​​su polusintetski, što općenito vrijedi za novije članove drugih skupina lijekova. Stabilnost tetraciklina u otopini ovisi o pH i derivatu. Lijekovi su amfoterni, što znači da će tvoriti soli i s jakim kiselinama i s bazama. Stoga mogu postojati kao soli natrija ili klorida.

Ne postoje krute podskupine tetraciklina, ali dok proučavate ovaj materijal, mogli biste primijetiti koliko ih često njihove karakteristike svrstavaju u jedan od 3 razreda u nastavku. One se temelje na dozi i učestalosti oralne primjene. Grupa 1 uključuje starije derivate kao što su klortetraciklin (sada se malo koristi), oksitetraciklin i tetraciklin. Grupa 2 uključuje demeklociklin i metaciklin. Grupa 3 uključuje novije lijekove kao što su doksiciklin i minociklin .

Mehanizam djelovanja

Tetraciklini se reverzibilno vežu na male podjedinice bakterijskih (i eukariotskih) ribosoma gdje ometaju vezanje nabijene tRNA na "prihvatljivo" mjesto. Oni su "bakteriostatski" prije nego cidali. Tetraciklini također mogu inhibirati sintezu proteina u domaćinu, ali je manja vjerojatnost da će postići potrebnu koncentraciju jer eukariotske stanice nemaju mehanizam preuzimanja tetraciklina.

Otpornost

S tetraciklinima se sve više susreću rezistentni organizmi kada se koriste u kliničkoj praksi, ali se još uvijek smatraju korisnima. Osjetljivi organizmi akumuliraju tetracikline intracelularno zbog aktivnog transportnog sustava. Nema poznatih enzima koji inaktiviraju tetracikline. Rezistencija na jedan tetraciklin obično implicira otpornost na druge, iako su neke istraživačke studije uočile razlike u MIC-ima za različite parove derivata tetraciklina - izolata. Te razlike nisu velike i nisu ujednačene u cijeloj zemlji.

Otpornost se prenosi u plazmidima koji kodiraju proteine ​​koji "ispumpavaju" lijekove iz stanica. Unutarstanična koncentracija predstavlja ravnotežu između ulaznih i izlaznih mehanizama. Postoji konceptualna sličnost u ovom mehanizmu rezistencije i onom kod stanica raka koje razvijaju otpornost na različite lijekove protiv raka u jednom koraku.

Farmakokinetika

Obratite pažnju na sličnost farmakokinetike doksiciklina i minociklina u raspravi koja slijedi. To su relativno novi tetraciklini koji su razvijeni za prevladavanje nedostataka u starijim derivatima.

Apsorpcija/primjena

Tetraciklini se prvenstveno koriste za oralnu primjenu, ali postoje lokalni, IM i IV oblici. Samo oksitetraciklin i tetraciklin imaju IM oblike doze, ostali uzrokuju sterilne apscese. IV injekcije se daju infuzijom kako bi se izbjegao kardiovaskularni kolaps. IV oblici doze postoje za minociklin, doksiciklin i dva koja također imaju oblike doze IM, oksitetraciklin i tetraciklin.

Tetraciklini se uvelike razlikuju u svojoj bioraspoloživosti i učinku koji hrana ima na nju. Doksiciklin i minociklin imaju vrlo visoku bioraspoloživost, u rasponu od 90 do 100%, a prisutnost hrane ima neznatan učinak. Ostali imaju bioraspoloživost od približno 58 do 77% i značajno se smanjuju hranom. Kalcij, aluminij i magnezij tvore netopive kelate s tetraciklinima kako bi smanjili bioraspoloživost. Mlijeko je bogato kalcijem, a svi ti ioni bogati su antacidima pa ih treba izbjegavati. Neki laksativi imaju magnezij. Budući da su tetraciklini iritansi koji izazivaju želučane smetnje, pacijente treba upozoriti da ne koriste mlijeko ili antacide za suzbijanje tegobe. Na sličan način treba upozoriti i vlasnike životinja.

Distribucija

Doksiciklin postiže terapeutske koncentracije u oku. Minociklin je također široko rasprostranjen, dostižući visoke koncentracije u slini i suzama. Oba se koriste u liječenju infekcija genitourinarnog trakta jer proizvode terapeutske koncentracije u tim tkivima, uključujući prostatu. Svi tetraciklini se distribuiraju u većinu tjelesnih tekućina uključujući transcelularne tekućine kao što su žuč, sinusne sekrecije, sinovijalne i pleuralne tekućine. Koncentracije u likvoru su 10-25% koncentracija u plazmi. To je dovoljno nisko da se ne preporučuju za infekcije CNS-a.

Radeći s govedom, Ziv i Sulman (1974.) otkrili su da je bilo potrebno približno 20 minuta da intravenozno primijenjeni doksiciklin i minociklin postignu omjer mlijeko:serum veći od 1,5. Tetraciklinu i oksitetraciklinu trebalo je 60 minuta da postignu omjer od 1,25 odnosno 0,75. Ove vrijednosti odražavaju razlike u sposobnosti lijekova da prođu kroz membrane koje su gore navedene. Međutim, imajte na umu da se u svim slučajevima omjer približio 1 ili više, što se ne vidi kod lijekova kao što su beta-laktami ili aminoglikozidi.

Tetraciklini imaju velike prividne volumene distribucije, u rasponu od 0,7 L/kg za doksiciklin do čak 1,9 za oksitetraciklin. Tetraciklini tipiziraju složenost korištenja Vd kao pokazatelja terapijskih koncentracija u tkivima. Tetraciklini se lokaliziraju u kostima, zubima, jetri, spini i tumorima. Budući da su jako vezani za ta tkiva i kosti, nehomogeno su raspoređeni izvan plazme. Paradoksalno, doksiciklin i minociklin prelaze membrane lakše nego bilo koji drugi, ali budući da visoko vezanje na proteine ​​plazme nadoknađuje nakupljanje u kostima i drugim tkivima, imaju Vds od 0,14 do 0,7 L/kg.

Volumen distribucije za životinje je u istom rasponu kao i za ljude, ali postoje značajne razlike. Na primjer, Vd minociklina je 1,9 L/kg kod pasa u odnosu na 0,4 za ljude. Vds oksitetraciklina su 1,4, 0,8, 2,1 i 2,1 L/kg za konje, goveda, pse i mačke. Imajte na umu da vrijednost za ljude, od 0,9 do 1,9 L/kg, zagrađuje raspon za ove vrste.

Eliminacija

Svi tetraciklini se eliminiraju putem bubrežnih i bilijarnih puteva, ali se razlikuju po relativnoj ovisnosti o njima. Svi prolaze kroz značajnu enterohepatičnu cirkulaciju. Doksiciklin i minociklin se primarno eliminiraju u žuči, a manje od trećine se eliminira nepromijenjeno. Oksitetraciklin, tetraciklin, metaciklin i demeklociklin eliminiraju se prvenstveno u urinu, a 42 do 70%, ovisno o derivatu, eliminira se u nepromijenjenom obliku.

Poluvijek eliminacije kreće se od 6-11 sati za tetraciklin i oksitetraciklin do 11 do 23 sati za doksiciklin i minociklin. Anurija gotovo ne mijenja brzinu eliminacije doksiciklina i minociklina, ali poluvrijeme eliminacije tetraciklina povećava se na 57 do 108 sati.

Poluvrijeme eliminacije oksitetraciklina, tetraciklina i minociklina obično su kraće u pasa, otprilike 6 sati, nego u ljudi (9,5, 10,6 i 17,5 sati). Konji i goveda imaju poluživot eliminacije oksitetraciklina sličan onima kod ljudi. [Konji/magarci mogu imati dulje poluživote što rezultira toksičnošću koja se često javlja, Bowersock, T. 1995.]

Gore navedeni podaci za doksiciklin i minociklin impliciraju da se oni u određenoj mjeri biotransformiraju u jetri. Doista, fenitoin ili barbiturat indukcija enzima koji metaboliziraju lijekove u jetri može smanjiti poluvijek eliminacije doksiciklina za više od 50%.

Štetni učinci

Tetraciklini se općenito smatraju relativno netoksičnima, ali izazivaju prilično velik broj štetnih učinaka, od kojih neki mogu biti opasni po život pod pravim okolnostima. Stoga ih ne treba koristiti slučajno.

Preosjetljivost

Alergijske reakcije nisu veliki problem s tetraciklinima iako se javljaju.

Biološki štetni učinci

Superinfekcija

Superinfekcija (suprainfekcija) može se pojaviti kod tetraciklina, osobito starijih, slabije apsorbiranih kada se daju oralno. Zbog svog širokog spektra djelovanja, djelovanja protiv komenzalnih organizama crijeva i učinkovite koncentracije u crijevima, gotovo uvijek mijenjaju crijevnu floru. To se može dogoditi unutar 24 do 48 sati, ali te promjene nisu uvijek klinički vidljive kao proljev. Nije neobično pronaći superinfekciju s kvascima ili otpornim patogenim bakterijama. Iako ih FDA ne voli, pripremljeni su komercijalni pripravci tetraciklina u kombinaciji s nistatinom (oralnim antifungalnim lijekom) kako bi pomogli u borbi protiv superinfekcije kvascima. Mnogi autoriteti vjeruju da, budući da se takve superinfekcije ne događaju uvijek, postoji manji rizik za pacijenta ako se čeka dok se ne pojave dokazi superinfekcije kvasca prije početka terapije.

Proljev

Može doći do proljeva koji će obično biti posljedica promjene mikroflore crijeva. Vidi raspravu o superinfekciji.

Probavne smetnje

Probavne smetnje mogu se pojaviti iz razloga koji su već navedeni pod naslovom superinfekcije. Može biti teško razlikovati probavne smetnje zbog promjena u flori od one uzrokovane izravnom iritacijom gastrointestinalne sluznice.

Probavne smetnje su potencijalno problematične kod preživača zbog velikog broja bakterija i protozoa u buragu. Konji, zečevi i druge životinje s velikom mikroflornom populacijom cekuma/debelog crijeva također su osjetljivi na učinke tetraciklina.

Bol u ustima i perinealni svrbež

Bol u ustima i perinealni svrbež zbog prekomjernog rasta gljivica su "češći" prema USPDI11th90.

Izravna toksičnost

Većina izravne toksičnosti posljedica je iritirajućih svojstava lijekova, inhibicije sinteze proteina ili njihove sklonosti koštanom tkivu.

Iritacija

Iritacija želučane sluznice koja dovodi do grčeva ili pečenja u želucu može biti takve jakosti da uzrokuje lošu suradljivost pacijenta. To često rezultira mučninom i povraćanjem. Imajte na umu da se minociklin i doksiciklin mogu uzimati s hranom kako bi se smanjio učinak ove iritacije.

Ista iritirajuća svojstva također ograničavaju upotrebu ovih lijekova za IM ili SC injekcije gdje svi uzrokuju bol, a većina uzrokuje sterilne apscese.

Taloženje u kalcificiranim tkivima

Taloženje u kalcificiranim tkivima, npr. zubima, može dovesti do promjene boje, osobito kada se daje tijekom razvojnih faza. Veće doze dane u neprikladnim fazama rasta mogu rezultirati deformacijom kosti. Gotovo svi koji su u djetinjstvu primali tetracikline imat će zube koji fluoresciraju pod UV svjetlom, bez obzira da li su im zubi obojeni u smeđu boju ili ne.

Vrtoglavica/lagana vrtoglavica

Vrtoglavica / vrtoglavica se obično viđa kod minociklina, ali ne i kod ostalih. To je uzrokovano vestibularnom ili CNS toksičnošću te je takve ozbiljnosti i učestalosti da je CDC promijenio preporuke o njegovoj nebitnoj uporabi.

Antianabolički učinak

Antianabolički učinak koji je posljedica smanjene sinteze proteina. U prisutnosti smanjene bubrežne funkcije to je očito kao azotemija i povećani dušik uree u serumu (SUN).

Fotoosjetljivost

fotoosjetljivost može biti povezana s primjenom svih tetraciklina, ali je posebno problem s demeklociklinom. Bolesnike treba držati podalje od jakog sunčevog svjetla kada primaju tetracikline.

Klinička primjena

Popis bolesti za koje se tetraciklini mogu koristiti je podugačak, ali zbog povećanja otpornosti postaje sve kraći. Preporuča se čitatelju da pogleda "trenutnu terapiju", udžbenik "medicine" ili referencu kao što je USPDI kako bi vidio raspon i vrste infekcija za koje se smatraju učinkovitom terapijom. Budući da su učinkoviti protiv širokog spektra bakterija i mnogih protozoa, njihova je primjena šira od mnogih antibakterijskih lijekova.

Tetraciklini su učinkoviti kod mnogih infekcija uzrokovanih gram-negativnim i gram-pozitivnim bakterijama. Primjeri uključuju Brucella , Francisella , Pseudomonas pseudomallei , Neisseria gonorrhea i Treponema pallidum .

Mnoge vrste Pasteurellae i Borrelia hurgdorferi (lajmska bolest). Najčešća upotreba u veterinarskoj medicini je u kombinaciji sa sulfama (npr. sulfadimetoksin [Albon] s kojim su sinergijski. Koristi se za liječenje većine infekcija Strept, Staph, Pasteurella kod goveda [Bowersock 1995).

Osim toga, tetraciklini su učinkoviti u rikecijalnim infekcijama, kao što su Q groznica i Stjenovita pjegava groznica, kao i one uzrokovane mikoplazmom i klamidijom. Posljednja dva često su uzroci upale pluća i infekcija genitourinarnog trakta. Psitakoza, uzrokovana Chlamydia psittac i, liječi se tetraciklinima.

Problematični slučajevi malarije i ameobiaze mogu imati koristi od tetraciklina koji se daju u kombinaciji sa specifičnijom antiinfektivnom terapijom.

Demeklociklin se također može koristiti za liječenje neinfektivnog problema poznatog kao sindrom neodgovarajućeg (viška) antidiuretskog hormona (SIADH). Djeluje tako da inhibira reapsorpciju vode izazvanu ADH u bubrezima kako bi se inducirala diureza vode. Očigledno je da se ova diureza može smatrati štetnim učinkom kada se koristi kao antiinfektivno sredstvo.

Reference

  1. Ziv i Sulman, Am. J. Vet. Rez. 35:1197,1974.
  2. USPDI, 11. izdanje, 1991
  3. USPDI, 15. izdanje, 1995
  4. BM6th88, Huber, W.G., Tetraciklini, u Veterinary Pharmacology and Therapeutics, 6. izdanje, ur. Booth, N.H. i McDonald, L.E., Iowa State University Press, 1988.
  5. Rang, H.P. i M.M. Dale. Farmakologija, Churchill Livingstone, New York 1987., 30. poglavlje.
  6. Bowersock, T., 1995. Osobna komunikacija.

Studijska pitanja

1. Koja je glavna osnova za odabir jednog lijeka iz tetraciklinske skupine? Pod pretpostavkom da ste odgovorili na farmakokinetička svojstva, kako se to može pomiriti s činjenicom da se specifični tetraciklini često preporučuju za specifične infektivne procese?

2. Minociklin je prije bio preporučeni tretman za nositelje meningokoka, ali CDC u Atlanti to više ne preporučuje? Koja je specifična toksičnost povezana s minociklinom? Što promjena u ovoj preporuci implicira o omjerima troškova i koristi za neke primjene lijekova?

3. Po čemu se tetraciklini (i sulfonamidi koji će se kasnije proučavati) razlikuju od ostalih antibakterijskih sredstava u svom djelovanju na protozoe? Znati imenovati dvije protozojske bolesti za koje su tetraciklini razumni dio terapije.

4. Što mislite zašto se skupina lijekova općenito smatra neotrovnom kada izazivaju toliko štetnih učinaka?

5. Trebali biste biti u stanju prepoznati i raspravljati o osnovi svakog od štetnih učinaka tetraciklina, npr. superinfekcija, proljev i pojačano SUN. Trebali biste biti u mogućnosti navesti i raspravljati o najmanje dva reprezentativna učinka iz svake od dvije važne (za tetracikline) kategorije štetnih učinaka.

6. Kakva je unakrsna rezistencija bakterija na tetracikline u usporedbi s onom beta-laktama i aminoglikozida?

7. Koje posebne mjere opreza treba poduzeti s tetraciklinima kada se koriste P.O.? Koja dva očito nisu pogođena ovim problemom?

8. Zašto se mnogi tetraciklini nikad ne koriste IM ili SC?

9. Zašto je intravenska primjena tetraciklina opasna, unatoč činjenici da je jedno od važnih sredstava za korištenje? Imajte na umu da neke osobe vjeruju da je kelacija kalcija uzrok ove hipotenzije, ali to nije nužno točno. Dodavanje kalcijevih soli u infuzije ne smatra se dobrom praksom. Spora administracija je!

10. Objasni kako prividni Vd nekih tetraciklina može biti veći od ukupne vode u tijelu?

11. Kakav je učinak loše funkcije bubrega ili jetre na brzinu eliminacije tetraciklina. Navedite jedan koji se primarno eliminira putem bubrega i onaj koji se primarno eliminira putem žuči.

12. Kako istodobna primjena fenitoina ili fenobarbitala i tetraciklina poput doksiciklina može dovesti do neuspjeha lijeka?


OSTATCI U MESU I MESNIM PROIZVODIMA | Ostaci hrane i lijekova

S. Croubels,. D. Courtheyn, u Encyclopedia of Meat Sciences, 2004

Tetraciklini

Tetraciklini su skupina antibiotika izvorno dobivenih iz određenih Streptomyces spp. Glavni predstavnici tetraciklinske skupine dostupni za liječenje životinja koje proizvode hranu su tetraciklin, oksitetraciklin, klortetraciklin i doksiciklin. Tetraciklini, koji su antibiotici širokog spektra, koriste se za liječenje, na primjer, respiratornih bolesti goveda, ovaca, svinja i pilića, a mogu se davati parenteralno, oralno ili lokalno. Nakon primjene tetraciklina na životinje, vezani ostaci antibiotika mogu se naći u kostima zaklanih životinja čak i mjesecima nakon tretmana. Ovi vezani ostaci mogu eventualno dospjeti u lanac ishrane preko kontaminiranog mesa (mehanički otkoštenog mesa) ili mesnog i koštanog brašna.

Kemijska struktura tetraciklina prikazana je u Slika 10 .


Tetraciklinski antibiotici: način djelovanja, primjena, molekularna biologija i epidemiologija rezistencije bakterija

Tetraciklini su otkriveni 1940-ih i pokazali su djelovanje protiv širokog spektra mikroorganizama uključujući gram-pozitivne i gram-negativne bakterije, klamidije, mikoplazme, rikecije i protozojske parazite. Oni su jeftini antibiotici, koji se intenzivno koriste u profilaksi i terapiji infekcija ljudi i životinja te također na subterapijskim razinama u stočnoj hrani kao promotori rasta. Prva bakterija otporna na tetraciklin, Shigella dysenteriae, izolirana je 1953. Rezistencija na tetraciklin sada se javlja u sve većem broju patogenih, oportunističkih i komenzalnih bakterija. Prisutnost patogena rezistentnih na tetraciklin ograničava upotrebu ovih sredstava u liječenju bolesti. Otpornost na tetracikline često je posljedica stjecanja novih gena, koji kodiraju za energetski ovisan efluks tetraciklina ili za protein koji štiti bakterijske ribosome od djelovanja tetraciklina. Mnogi od ovih gena povezani su s mobilnim plazmidima ili transpozonima i mogu se međusobno razlikovati korištenjem molekularnih metoda uključujući hibridizaciju DNA-DNA s oligonukleotidnim sondama i sekvenciranje DNA. Ograničeni broj bakterija stječe otpornost mutacijama, koje mijenjaju propusnost porina vanjske membrane i/ili lipopolisaharida u vanjskoj membrani, mijenjaju regulaciju urođenih efluksnih sustava ili mijenjaju 16S rRNA. Novi derivati ​​tetraciklina se ispituju, iako njihova uloga u liječenju nije jasna. Promjena upotrebe tetraciklina u zdravlju ljudi i životinja kao i u proizvodnji hrane potrebna je ako želimo nastaviti koristiti ovu klasu antimikrobnih sredstava širokog spektra kroz današnje stoljeće.

Figure

Struktura 6-deoksi-6-demetiltetraciklina, minimalno…

Struktura 6-deoksi-6-demetiltetraciklina, minimalnog farmakofora tetraciklina.

Stereokemijski i zamjenski zahtjevi za…

Stereokemijski i supstitucijski zahtjevi za optimalnu antibakterijsku aktivnost unutar serije tetraciklina.


Mjere opreza

Općenito

Kao i kod drugih antibakterijskih lijekova, uporaba ovog lijeka može dovesti do prekomjernog rasta neosjetljivih organizama, uključujući gljivice. Ako dođe do superinfekcije, prekinuti antibakterijsku terapiju i uvesti odgovarajuću terapiju.

Liječite sve infekcije uzrokovane beta-hemolitičkim streptokokom grupe A najmanje deset dana.

Izvedite inciziju i drenažu ili druge kirurške zahvate zajedno s antibakterijskom terapijom, kada je to indicirano.

Malo je vjerojatno da će propisivanje tetraciklina u nedostatku dokazane ili jako sumnjive bakterijske infekcije ili profilaktičke indikacije donijeti korist za bolesnika i povećava rizik od razvoja bakterija rezistentnih na lijekove.

Informacije za pacijente

Savjetujte pacijente da se antibakterijski lijekovi uključujući tetraciklin smiju koristiti samo za liječenje bakterijskih infekcija. Ne liječe virusne infekcije (npr. prehladu). Kada se tetraciklin propisuje za liječenje bakterijske infekcije, recite pacijentima da, iako je uobičajeno da se osjećaju bolje na početku terapije, lijek treba uzimati točno prema uputama. Preskakanje doza ili nedovršavanje cijelog tijeka terapije može (1) smanjiti učinkovitost neposrednog liječenja i (2) povećati vjerojatnost da će bakterije razviti otpornost i da se neće moći liječiti tetraciklinom ili drugim antibakterijskim lijekovima u budućnosti.

Laboratorijski testovi

Kod spolno prenosivih infekcija, kada se sumnja na koegzistentni sifilis, obavite tamne preglede prije početka liječenja i ponavljajte serologiju krvi svaki mjesec najmanje četiri mjeseca.

Interakcije s lijekovima

Budući da bakteriostatski lijekovi mogu utjecati na baktericidno djelovanje penicilina, preporučljivo je izbjegavati davanje tetraciklina u kombinaciji s penicilinom ili drugim baktericidnim antibakterijama.

Budući da se pokazalo da tetraciklini smanjuju aktivnost protrombina u plazmi, pacijenti koji su na terapiji antikoagulansima mogu zahtijevati smanjenje doze antikoagulansa.

Zabilježeno je da istodobna primjena tetraciklina i metoksiflurana dovodi do smrtonosne bubrežne toksičnosti.

Apsorpciju tetraciklina smanjuju antacidi koji sadrže aluminij, kalcij ili magnezij i pripravci koji sadrže željezo, cink ili natrijev bikarbonat.

Istodobna primjena tetraciklina može učiniti oralne kontraceptive manje učinkovitima.

Karcinogeneza, mutageneza, oštećenje plodnosti

Trenutno se provode dugoročne studije na životinjama kako bi se utvrdilo ima li tetraciklin hidroklorid karcinogeni potencijal. Neki srodni antibakterijski lijekovi (oksitetraciklin, minociklin) pokazali su dokaze onkogenog djelovanja u štakora.

U dva in vitro sustava ispitivanja stanica sisavaca (L 51784y limfom miša i stanice pluća kineskog hrčka), postojali su dokazi o mutagenosti s tetraciklin hidrokloridom.

Tetraciklin hidroklorid nije imao utjecaja na plodnost kada se davao u prehrani mužjacima i ženkama štakora uz dnevni unos od približno 400 mg/kg/dan, što je otprilike 8 puta više od najveće preporučene doze za ljude na temelju tjelesne površine.

Trudnoća

Trudnice s bubrežnom bolešću mogu biti sklonije razvoju zatajenja jetre povezanog s tetraciklinom.

Rad i dostava

Učinak tetraciklina na trudove i porođaj nije poznat.

Dojilje

Zbog mogućnosti ozbiljne nuspojave kod dojenčadi od tetraciklina, treba donijeti odluku hoće li se lijek prekinuti, uzimajući u obzir važnost lijeka za majku (vidi UPOZORENJA).

Pedijatrijska uporaba


Antimikrobno djelovanje

Način djelovanja:

Antimikrobna aktivnost tetraciklina odražava reverzibilno vezanje za bakterijsku 30S ribosomsku podjedinicu, a posebno na mjesto akceptora aminoacil-tRNA ("quotA") na mRNA ribosomskom kompleksu, čime se sprječava ribosomalna translacija. Taj je učinak također očit u stanicama sisavaca, iako su mikrobne stanice selektivno osjetljivije zbog većih koncentracija. Tetraciklini ulaze u mikroorganizme djelomično difuzijom, a dijelom putem energetski ovisnog sustava posredovanog prijenosnikom koji je odgovoran za visoke koncentracije koje se postižu u osjetljivim bakterijama. Tetraciklini su općenito bakteriostatski, a sustav obrane domaćina koji reagira neophodan je za njihovu uspješnu upotrebu. U visokim koncentracijama, koje se mogu postići u urinu, postaju baktericidne jer se čini da organizmi gube funkcionalni integritet citoplazmatske membrane. Tetraciklini su učinkovitiji protiv mikroorganizama koji se razmnožavaju i imaju tendenciju da budu aktivniji pri pH 6-6,5. Antibakterijska učinkovitost opisana je kao ovisna o vremenu.

Otpornost na bakterije:

Najčešći mehanizam kojim mikrobi postaju rezistentni na tetracikline je smanjeno nakupljanje lijeka u prethodno osjetljivim organizmima. Dva mehanizma uključuju 1) poremećenu apsorpciju u bakterije, što se događa kod mutantnih sojeva koji nemaju potreban transportni sustav, i 2) mnogo češće stjecanje aktivnih efluksnih pumpi posredovano plazmidom ili transpozonom. Genomi za ove sposobnosti mogu se prenijeti ili transdukcijom (kao u Staphylococcus aureus) ili konjugacijom (kao kod mnogih enterobakterija). Drugi mehanizam rezistencije je proizvodnja "zaštitnog" proteina koji djeluje ili sprječavajući vezanje, uklanjajući vezani lijek ili mijenjajući negativan utjecaj vezanja na ribosomalnu funkciju. Među tetraciklinima, tigeciklin karakterizira manja otpornost zbog efluksa ili zaštite ribosoma. Rijetko se tetraciklini mogu uništiti acetilacijom. Rezistencija se polako razvija u više koraka, ali je široko rasprostranjena zbog široke upotrebe niskih koncentracija tetraciklina.

Antimikrobni spektri:

Svi tetraciklini su približno jednako aktivni i obično imaju približno isti široki spektar, koji uključuje i aerobne i anaerobne gram-pozitivne i gram-negativne bakterije, mikoplazme, rikecije, klamidije, pa čak i neke protozoe (amebe). Tetraciklini su općenito lijek izbora za liječenje rikecija i mikoplazme. Među osjetljivim organizmima je Wolbachia, intracelularni endosimbiont nematoda sličan rikecijama, uključujući Dirofilaria immitis. Sojevi od Pseudomonas aeruginosa, Proteus, Serratia, Klebsiella, i Trueperella spp često su otporni, kao i mnogi patogeni Escherichia coli izolira. Iako postoji opća unakrsna rezistencija među tetraciklinima, doksiciklin i minociklin obično su učinkovitiji protiv stafilokoka.


Izgledi za budućnost

Veza između primjene antibiotika i razvoja bakterijske rezistencije utvrđena je tijekom posljednjih 60 godina. Koliko brzo određeni patogen stječe rezistenciju na tetraciklin ovisi o brojnim čimbenicima, od kojih mnogi ostaju slabo definirani. Nekoliko novih tetraciklinskih spojeva se ispituje ili su u kliničkim ispitivanjima, ali je malo vjerojatno da će u bliskoj budućnosti biti dostupno mnogo više derivata. Nažalost, bakterije ne razlikuju upotrebu u liječenju bakterijske infekcije od upotrebe u liječenju neinfektivnih stanja. To je zabrinjavajuće jer je primjena tetraciklina mnogo šira nego prije 20-30 godina. Globalna potrošnja tetraciklina godišnje nije poznata. Može se pretpostaviti da će se tetraciklini sve više koristiti protiv protozojskih parazitskih bolesti i možda drugih parazitskih bolesti u budućnosti. Uloga antibiotika u proizvodnji hrane i utjecaj toga na otpornost bakterija ljudskih patogena bila je tema od velikog interesa, a nedavni dodatak Kliničke zarazne bolesti [24] ide u detalje o toj temi. Možda najveću zabrinutost izaziva dugotrajna subterapijska uporaba tetraciklina za liječenje nebakterijskih neinfektivnih stanja. Ova vrsta niske razine, dugotrajna uporaba stavlja značajan selektivni pritisak na bakterije koje nosi domaćin i u okoliš domaćina koji se liječi. Ako se ništa ne poduzme, korisnost tetraciklina kao antibakterijskog sredstva je ograničena kako raste otpornost bakterija. Stoga moramo smanjiti sve vrste uporabe ovog sredstva i svih antibiotika koji se koriste u cijelom svijetu ako se nadamo da ćemo tetraciklinsku terapiju zadržati kao opciju za ovo stoljeće.

Konačno, biološki terorizam je tijekom protekle godine postao stvarna prijetnja. Sugerirano je da bi najvjerojatnije bakterijsko oružje bilo Bacillus anthracis, Francisella tularensis, i/ili Yersinia pestis [11]. U svakom slučaju, doksiciklin je važan za liječenje i/ili profilaksu (tablica 1). Nažalost, otporan na tetraciklin Y. pestis već je opisano, ali je rijetko [1]. However, last year, during the anthrax attacks, the general public accumulated antibiotics at home and often took these antibiotics in anticipation of exposure without doctor's consultation [ 11]. This unnecessary exposure to antibiotics by large numbers of people may lead to increased prevalence of antibiotic-resistant bacteria in the community, both for the 3 agents that could be used in biological weapons and for any other pathogenic bacteria. Thus, education of the public, health authorities, and clinicians is needed now more than ever to eliminate home stockpiling of antibiotics, to ensure the correct use for all bacterial infections (especially during a biological attack), and to prevent use when antibiotics are not needed, such as use for treatment of viral infections.


Tetracycline Antibiotics

Tetracycline antibiotics used clinically include doxycycline, minocycline, and tetracycline, while tigecycline, which has the same four-ringed structure and is a derivative of minocycline, is the first member of the glycylcyclines to be approved their structures are shown in Figure 4.3.1 and their therapeutic indications are listed in Table 4.3.1.

Table 4.3.1 Therapeutic indications for the tetracycline antibiotics.

























Tetracycline antibiotic Indikacije
Doxycycline Chronic prostatitis, sinusitis, syphilis, uncomplicated genital chlamydial infection, pelvic inflammatory disease, acne vulgaris, rosacea, Lyme disease, community-acquired pneumonia
Lymecycline Acne vulgaris
Minocycline Acne vulgaris, prophylaxis of asymptomatic meningococcal carrier state (no longer recommended)
Oxytetracycline Acne vulgaris, rosacea
Tetraciklin Acne vulgaris, rosacea, non-gonococcal urethritis, chronic bronchitis
Tigecycline Complicated intra-abdominal or skin/soft tissue infections

The tetracycline antibiotics are the third example so far in this section, of naturally occurring molecules from a microbial source that interfere with bacterial protein synthesis. Once again, the first in this series, chlortetracycline (originally named aureomycin), resulted from a programme of screening soil microorganisms for potential new antibiotics. This discovery is attributed to Benjamin M. Duggar, a retired botanist, with expertise across a wide range of plant and microorganism physiology. He retired from Wisconsin University in 1943, when he was 71, but was approached to act as consultant for Lederle Laboratories in New York (part of American Cyanamid Company, now part of Wyeth Pharmaceuticals), who were supporting the war effort by searching for new antibiotic and antimalarial agents (Walker, 1982).

Although he is solely credited with the discovery of chlortetracycline in 1948, Duggar was part of a larger team, under the direction of Yellapragada SubbaRow, which was systematically investigating soil microorganisms for natural products with desirable pharmaceutical activities. Duggar requested some local samples from the soil microbiologist at the University of Missouri, William Albrecht, from which he cultured a golden mould, which produced a yellow pigment that displayed growth inhibitory properties against bacteria, such as streptococci. He identified the mould as a Streptomyces species that had not previously been catalogued to reflect its colour, he named it Streptomyces aureofaciens and the antibiotic it produced aureomycin (Duggar, 1948). Note that the ‘mycin’ part of the name corresponds with its isolation from a Streptomyces species, like the aminoglycoside streptomycin and the macrolide erythromycin, which we have just met.

Aureomycin was quickly released to clinicians and other researchers to obtain evaluative data of its activity and efficacy it gathered support with glowing testimonials of its broad spectrum of activity, including against streptomycin- and penicillin-resistant organisms (Wright and Schreiber, 1949 Cantor, 1950 Kiser et al ., 1952). It was found to be as effective as penicillin and streptomycin and had the significant advantage of being the first antibiotic that was effective when administered orally.

Following the discovery of aureomycin, other tetracyclines were soon discovered:


  • Oxytetracycline (originally called terramycin) in 1949 from Streptomyces rimosus by Pfizer (Finlay et al ., 1950).
  • Tetracycline (marketed originally as achromycin (Darken et al ., 1960)) in 1953 from S. aureofaciens when cultured with a chlorination inhibitor (Goodman and Matrishin, 1968).
  • Demethylchlortetracycline in 1957 from S. aureofaciens (McCormick et al ., 1957 Wilson, 1961). This was the last natural tetracycline to be identified and was originally called declomycin or ledermycin. It is still marketed as the latter (or under its generic name, demeclocycline) by Lederle Laboratories.

The discovery of this new class of antibiotics is not without controversy, though: Pfizer, American Cyanamid, and Bristol-Myers formed a monopoly that maintained artificially high prices for tetracycline over several years before the US Federal Trade Commission halted the violations after a series of high-profile investigations, charges, and appeals heard in the high court (Anon, 1964 US Court of Appeals, 1968).

When it was discovered that the hydrogenation of chlortetracycline resulted in dechlorination and conversion into tetracycline (which was as active as chlortetracycline) (Stephens et al ., 1952 Conover, 1955), the possibility that synthetic modification of tetracyclines might provide alternative agents with antibacterial activity was realised. During the next 15–20 years, many semi-synthetic analogues were prepared, including lymecycline, doxycycline, and minocycline some of these second-generation semi-synthetic tetracyclines were even more potent than chlortetracycline and are still marketed today. Structural modification has continued and has resulted in the discovery of a third-generation tetracycline, t -butylglycylamidominocycline (tigecycline, originally labelled GAR-936) (Petersen et al ., 1999), with more in development and in clinical trials (Sun et al ., 2008 Brötz-Oesterhelt and Sass, 2010).

Structurally, the tetracyclines are based on a four-ring (tetracyclic or octahydronaphthacene) system, hence the name the rings are labelled A, B, C, and D (Figure 4.3.2). One face consists of carbonyl, phenol, alcohol, and enol oxygen atoms, with high polarity and metal ion binding ability, while the other face is substantially less polar. There are a number of substitution patterns commonly found in the antibiotic tetracyclines and significant deviation from these leads to greatly reduced antibacterial activity (Chopra and Roberts, 2001 Zhanel et al ., 2004).

Figure 4.3.2 Requirements for tetracycline antibiotic activity (Chopra and Roberts, 2001 Zhanel et al ., 2004)

Much of the development of new tetracycline antibiotics has been driven by the instability of the first-generation tetracyclines, particularly chlortetracycline, oxytetracycline, and tetracycline, which can lead to degradation during storage and even production of a toxic product. We will look at some of the reactions of tetracyclines and the resultant effects upon bioavailability in Subsection 4.3.3.

Looking back at Figure 4.3.1, you can see that the tetracyclines have a number of chiral centres and functional groups, so you will not be surprised to learn that fermentation methods are considered to be the most cost-effective for their production, and for the production of the base structures for semi-synthetic analogues, such as lymecycline and tigecycline (Khosla and Tang, 2005). As you will see further on in this subsection, there is now an efficient chemical synthetic method for multigram quantities of a key intermediate in tetracycline synthesis, which offers the possibility of analogue synthesis (Brubaker and Myers, 2007). The first patented fermentations of S. aureofaciens were for the production of chlortetracycline (Duggar, 1948 Neidercorn, 1952) and tetracycline (Goodman et al ., 1959) much work since then has focussed on optimising the selectivity for, and the yields of, the desired tetracyclines, especially since some Streptomyces species can produce more than one tetracycline, depending upon the fermentation conditions (Bêhal, 1987, 2000). In early 2011, there were almost 3000 patents relating to the biosynthesis and synthesis of tetracycline and its analogues ( worldwide.espacenet.com ), including some filed in 2010 and 2011 – proof that there is still interest in the production and use of tetracyclines. It should be noted, however, that some of these were for non-antibiotic uses of tetracyclines, briefly mentioned in Subsection 4.3.4.

4.3.2.1 Biosynthesis of Tetracyclines

The biosynthesis of tetracycline antibiotics is related to the bacterial synthesis of fatty acids through the bacterial type II polyketide synthase pathway, consisting of a well-studied set of enzymes (for examples, see Khosla, 2009 Zhang and Tang, 2009), although the synthesis of tetracyclines is unique to certain bacteria (Clardy et al ., 2009). The biosynthetic pathways to tetracycline and oxytetracycline are the most studied (for example, Petkovi et al ., 2006 Pickens and Tang, 2009). The biosynthetic pathway to natural tetracyclines is available from the KEGG database ( www.genome.jp/kegg/pathway/map/map00253.html , last accessed 10 March 2012) in summary, it uses the precursor, malonamyl coenzyme A (CoA), which is obtained from acetyl CoA via malonyl CoA and glutamine (Wang et al ., 1986), and proceeds through two common key intermediates, 6-methylpretetramide and 4-ketoanhydrotetracycline (Scheme 4.3.1 and Table 4.3.2) (Clardy et al ., 2009 Pickens and Tang, 2009).

Scheme 4.3.1 Biosynthetic pathway to tetracycline antibiotics (Clardy et al ., 2009 www.genome.jp/kegg/pathway/map/map00253.html , last accessed 10 March 2012)

Table 4.3.2 Enzymes involved in tetracycline biosynthesis.














































Enzim Funkcija
OxyA Ketosynthase
OxyB Chain length factor
OxyC Acyl carrier protein
OxyJ Ketoreductase
OxyK Aromataza
OxyN Cyclase
OxyF C-methyltransferase
OxyE Flavin-dependent monoxygenase
OxyL NADPH-dependent dioxygenase
OxyQ Aminotransferase
OxyT N,N -dimethyltransferase
OxyS Monooxygenase that hydroxylates stereospecifically at C6
Cts4 Halogenase

For clarity, in Scheme 4.3.1 the precursor unit, malonamyl CoA, is coloured pink throughout the consecutive acetyl units added to malonamyl CoA from acetyl CoA are coloured alternately black and red – you can see that the cycle of acetyl addition occurs eight times until the linear nonaketamide is formed. A series of enzymic reactions involving OxyJ, OxyK, and OxyN leads to pretetramide (not shown in Scheme 4.3.1), which is converted into 6-methylpretetramide by OxyF. This series of reactions results in cyclisation of the nonaketamide to the tetracyclic structure of 6-methylpretetramide, followed by methylation at C6, with the new bonds formed in this sequence shown in blue. If you trace the sequentially added acetyl units, you can see how the two-carbon units form the backbone of the structure. Oxidation at C4 (by OxyE), and hydroxylation at C12a by OxyL, provides the second key intermediate, 4-ketoanhydrotetracycline, at which the tetracycline biosynthetic paths diverge. Chlortetracycline results from Cts4 halogenase action at C7 (Dairi et al ., 1995), followed by amination at C4 (OxyQ) and OxyT N , N -dimethylation (the methyl groups are provided by S -adenosyl methionine) OxyS catalyses the stereospecific hydroxylation at C6 , leaving a stereospecific reduction at C5a required to produce chlortetracycline. In the other pathway, amination at C4 , dimethylation , and hydroxylation at C6 provide 5a,11a-dehydrotetracycline, from which oxytetracycline and tetracycline are obtained.

Much detailed research has been directed at elucidating this pathway most was carried out on the enzymes of the oxytetracycline-producing species Streptomyces rimosus , hence the Oxy names, but the enzymes are the same or similar for the other tetracycline-producing species (Zhang et al ., 2007 Petkovi et al ., 2010 Pickens and Tang, 2010).

4.3.2.2 Chemical Synthesis of Tetracyclines

The literature related to the chemical synthesis of tetracyclines resembles a ‘Who’s Who’ of synthetic organic chemistry:


  • R. B. Woodward solved the structure, complete with stereochemistry, in 1952 (this was revised slightly in the 1960s with the help of X-ray crystallography) (Hochstein et al ., 1953 Donohoe et al ., 1963 von Wittenau et al ., 1965).
  • Woodward and Conover (who first synthesised tetracycline by hydrogenation of chlortetracycline) synthesised a biologically active tetracycline, named sancycline (Korst et al ., 1968), albeit in 25 steps and 0.002% overall yield.
  • Shemyakin synthesised a tetracycline natural product, (±)-12a-deoxy-5a,6-anhydrotetracycline (Gurevich et al ., 1967).
  • Muxfeldt identified the major problems with the synthesis of tetracyclines: the complexity of the required stereochemistry and the sensitivity of the tetracycline functional groups to both mild acid and base during initial studies (Muxfeldt and Rogalski, 1965), then later achieved the total synthesis of (±)-5-oxytetracycline in 22 steps and 0.06% (Muxfeldt et al ., 1968, 1979).
  • Stork concentrated on achieving the correct stereochemistry at each centre in the basic tetracycline structure, producing (±)-12a-deoxytetracycline in 16 steps and an impressive 18–25% yield, although this structure has little antimicrobial activity (Stork et al ., 1996).
  • Tatsuta and co-workers exploited the natural stereochemical definition of carbohydrates for their starting materials and achieved the total synthesis of natural (−)-tetracycline from D-glucosamine in 34 steps and 0.002% yield (Tatsuta et al ., 2000 Tatsuka and Hosokawa, 2005), including a solution to the difficult stereospecific hydroxylation of C12a.
  • More recently, Myers and co-workers developed a highly effective synthetic approach to natural tetracyclines, their analogues, and their precursors (Charest et al ., 2005a, 2005b Brubaker and Myers, 2007 Myers et al ., 2007, 2011 Sun et al ., 2008), which takes account of the considerable challenge in achieving the correct stereochemistry, particularly at C12a, and provides versatility for the synthesis of many new analogues for microbiological evaluation (Myers et al ., 2007 Sun et al ., 2008).

A discussion of the chemical strategies for synthesising tetracyclines, the reactions required, and their stereochemical complexities would be a section in itself, so we will restrict ourselves here to consideration of the most recent syntheses, which have enabled multigram quantities of optically pure tetracyclines to be achieved and thus offer potential commercial synthetic routes to these agents. Myers and colleagues recognised that one key intermediate 1 , providing the A and B rings of tetracyclines, allows the synthesis of a wide range of tetracycline antibiotics and their analogues (Scheme 4.3.2).

Scheme 4.3.2 Key intermediates in the synthesis of tetracycline antibiotics (Myers et al ., 2007, 2011)

Initially, they developed a synthesis to this important intermediate from benzoic acid and achieved intermediate 1 in 21% overall yield after 7 steps (Charest et al ., 2005a). Using this route, (−)-tetracycline could be synthesised in 17 steps and 1.1% yield from benzoic acid. Conversion of 1 into 2 and 3 provided routes to tetracyclines with no hydroxyl group at C5 (including an alternative synthesis of tetracycline) and 5-hydroxytetracyclines, respectively. Using this approach, the synthesis of (−)-6-deoxytetracycline was achieved in 14 steps and 7% yield via intermediate 2 , while (−)-doxycycline was isolated in 8.3% yield after 18 steps via intermediate 3 the yields and number of steps relate to the total synthesis from benzoic acid (Charest et al ., 2005a, 2005b Myers et al ., 2007, 2011). Not content with these impressive achievements, Myers and Brubaker re-designed and improved the synthesis of intermediate 2 (Scheme 4.3.3), from which most tetracyclines can be accessed they identified an alternative cheap and readily available commercial starting material, methyl 3-hydroxy-5-isoxazolecarboxylate 4 , along with an improved synthetic strategy to improve stereoselectivity and yields, and obtained intermediate 2 in 9 steps and 21% overall yield from starting material 4 (Brubaker and Myers, 2007).

Scheme 4.3.3 Improved synthesis of key intermediate 2 for large-scale tetracycline synthesis (Brubaker and Myers, 2007)

There are several noteworthy features in this revised synthesis of intermediate 2 (Scheme 4.3.3):


  • First, the introduction of a stereogenic (chiral) centre into structure 5 is carried out in high yield and with a high enantiomeric excess, and this enantiomeric ratio is maintained during the S N 2 replacement of the hydroxyl group (as a mesylate) with a dimethylamino group in the synthesis of 6 .
  • The resulting stereogenic centre at C6 becomes C4 in the tetracycline and already has the correct stereochemistry, which is retained throughout the remainder of the synthesis.
  • Intermediate 7 has a new stereogenic centre bearing a hydroxyl group you may be concerned that the stereochemistry is not defined at this new centre, but this group is oxidised to a carbonyl during the synthetic sequence that results in intermediate 8 , so the mixed stereochemistry does not matter at this stage.

So far, we have not considered how rings C and D can be constructed, yet this is just as important for the tetracycline structure. The synthetic strategy adopted by the Myers group uses intermediate 1 , 2 , or 3 as appropriate to provide rings A and B of the tetracycline with the correct stereochemistry and functionality, then elaborates this basic structure by construction of the C-ring, while adding the D-ring through a generalised Michael–Dieckmann reaction sequence (using a carbanion formed from a variety of D-ring precursors), followed by deprotection of all the functional groups (Scheme 4.3.4) (Charest et al ., 2005a).

Scheme 4.3.4 Elaboration of intermediates 2 and 3 into tetracyclines and their analogues (Charest et al ., 2005a)

One great advantage of this approach is the ease with which varying functionality can be added throughout the structure, particularly substituents at C5 ( X ), C6 ( R ), C7 ( Y ), C9 ( Z ), and even an extra ( E ) ring a great many analogues have been made, and some of these combine strong antibacterial activity with activity against strains resistant to first- and second-generation tetracycline antibiotics (Myers et al ., 2011 Sun et al ., 2011). The synthetic routes to the tetracyclines developed by the Myers group have been sufficiently successful to support a spin-out company, Tetraphase, which has several tetracyclines in early clinical trials. Other groups have also pursued C9-substituted tetracyclines (for example, Koza and Nsiah, 2002 Sum et al ., 2006) the clinical success of tigecycline (see Subsections 4.3.4 and 4.3.5) and the development of amadacycline (which is in clinical trials and is discussed in Subsection 4.3.9) provided the rationale for their evaluation.

The pharmacokinetics and pharmacodynamics of the tetracycline antibiotics were reviewed recently (Agwuh and MacGowan, 2006 Barbour et al ., 2010), but they are not fully understood, with several seemingly contradictory observations. The tetracyclines display time-dependent effects, yet the general concentration-dependent parameters (of exposure time at a concentration above the MIC) provide good clinical results, with a strong post-antibiotic effect. Although generally considered to be bacteriostatic, there is evidence of bactericidal activity with certain tetracycline antibiotics against specific bacteria, when used at an appropriate concentration (Zhanel et al ., 2004 Barbour et al ., 2010).

The instability of the first-generation tetracyclines during storage has already been mentioned we will consider the reactions of tetracyclines more carefully here, as they have an effect upon bioavailability and even upon the safety of the products.

The first-generation tetracyclines, although clinically successful, were found to be unstable to acidic, basic, and neutral pH during storage and in solution, including in the gastrointestinal (GI) tract after administration, decreasing their bioavailability (Walton et al ., 1970 Ali and Strittmatter, 1978 Wu and Fassihi, 2005). Two main reactions occur in the presence of acid: epimerisation at C4, to produce epitetracycline (Hussar et al ., 1968) (Scheme 4.3.5), and dehydration of 6-hydroxytetracyclines across C5a-C6 (with loss of the OH group at C6), to give the anhydrotetracycline derivative, as demonstrated for tetracycline in Scheme 4.3.6.

Scheme 4.3.5 Acid-catalysed epimerisation at C4 of tetracycline

Scheme 4.3.6 Acid-catalysed dehydration of chlortetracycline and improved stability of demethylchlortetracycline (demeclocycline)

The dehydration (shown in the upper part of Scheme 4.3.6 for chlortetracycline) proceeds through an E1 mechanism, involving protonation of the C6-OH and loss of a good leaving group (H 2 O), to produce a stabilised tertiary carbocation at C6 , followed by loss of a proton from C5a to form the alkene group of anhydrotetracycline. The slightly improved acid stability of demeclocycline and slower dehydration is due to fact that the secondary carbocation that has to be formed at C6 is less stable, and this process thus has a greater activation energy. In this latter case, reversible protonation of the C6-OH favours demeclocycline, instead of proceeding to the carbocation (Scheme 4.3.6).

Epimerisation at C4 of the anhydrotetracycline derivatives can also occur, producing the corresponding epianhydrotetracycline derivative (Sokoloski et al ., 1977). Some of the products formed by acid- or base-catalysed degradation are themselves active as antibiotics, while others, for example anhydrotetracycline and epianhydrotetracycline, are toxic (Mull, 1966 Kunin, 1967). The adverse effects (which can manifest as Fanconi syndrome see Subsection 4.3.7) of using tetracyclines that have degraded during storage were observed soon after these agents had been adopted into regular use, but it was several more years before reliable analytical methods for their analysis and quality control were developed (Frimpter et al ., 1963 Gross, 1963 Butterfield et al ., 1973). We will discuss the adverse effects of tetracyclines in Subsection 4.3.7 our main concern here is that the bioavailability of some tetracyclines (particularly the first-generation members) can be reduced by their degradation during storage, particularly in solution (Wu and Fassihi, 2005), or as a result of GI-induced reactions (Okeke and Lamikanra, 1995 Dos Santos et al ., 1998). The second-generation tetracyclines, doxycycline and minocycline, and the third-generation, tigecycline, are more stable to acidic pH, as they do not have a C6-OH substituent to be protonated and eliminated as water.

Lymecycline is a prodrug form of tetracycline that is hydrolysed at acidic and neutral pH in vivo to tetracycline, formaldehyde (methanal), and the amino acid lysine after oral and parenteral administration (Scheme 4.3.7). Interestingly, it has lower oral bioavailability than the parent compound, tetracycline (Sjölin-Forsberg and Hermansson, 1984).

Scheme 4.3.7 In vivo hydrolysis of lymecycline to form tetracycline

The balance of lipophilicity to hydrophilicity in tetracyclines is affected by pH (Chen and Lin, 1998) such a property is usually a clue that there are functional groups in the molecule under investigation which are ionisable at physiological pH values. Each tetracycline has at least one readily ionisable amine group (at C4) and two enol groups (at C3 and C12) that are also relatively easily ionised. First- and second-generation tetracyclines have three pK a values in the physiological pH range (generally around 3.2, 7.6, and 9.6) – the two enol groups, at C3 and C12 , are the most acidic (and so have the lower pK a values). The pK a of the phenol group at C10 (

12) means that it is not ionised at physiological pH values. The acid–base equilibria for tetracycline, which are typical of the tetracycline antibiotics, are demonstrated in Scheme 4.3.8 (Jin et al ., 2007).

Scheme 4.3.8 Acid–base equilibria for tetracycline (Jin et al ., 2007)

The fully protonated form of the tetracyclines (with an overall charge of +1) is likely to predominate in the acidic medium of the stomach, minimising absorption from this compartment (as neutral species are absorbed best). It is not surprising to find that tetracycline antibiotics are absorbed chiefly from the duodenum, where the pH is around 6–6.5 and the overall neutral form of the tetracycline predominates (Colaizzi and Klink, 1969). By modifying the pH, greater aqueous solubility of the tetracycline antibiotics can be achieved, making them suitable for parenteral administration, and oxytetracycline, lymecycline, doxycycline, and minocycline have been formulated in this way (Chopra and Roberts, 2001).

The third-generation antibiotic tigecycline has limited oral bioavailability and is administered by IV infusion, due to its greater hydrophilicity and reduced lipophilicity as a result of its extra ionisable groups (Meagher et al ., 2005). Tigecycline has two extra ionisable groups (Figure 4.3.3):

Figure 4.3.3 Tigecycline pKa values (Tygacil_BioPharmr)


  • The t -butylamine on the side chain at C9 (a secondary aliphatic amine, so expected to be basic, with a pK a (of the conjugate acid, R 3 NH + ) of 8.5–10).
  • The dimethylamino group at C7 (an aromatic amine, so expected to be a weak base, due to resonance of the nitrogen lone pair with the π-system of the aromatic ring, with a pK a of 3.5–5).

Besides being affected by pH, the absorption of the first-generation tetracyclines in particular, and some of the second-generation analogues, is adversely affected by their concurrent administration with food (Schimdt and Dalhoff, 2002 Agwuh and MacGowan, 2006), dairy products, and other metal-ion-containing preparations (such as antacid treatments), although there is some evidence that the absorption of lymecycline is less affected by milk (Ericson and Gnarpe, 1979). Tetracycline has a logP value of 0.09 and a bioavailability of around 75%, which is reduced by about 50% when co-administered with food (Miller et al ., 1977 Zhanel et al ., 2004). By comparison, doxycycline and minocycline have a greater bioavailability (90–100%) (Zhanel et al ., 2004), their absorption is not affected by food, and they are well absorbed after oral administration. The greater lipophilicity of these agents (logP values of 0.95 and 1.12, respectively) (Colaizzi and Klink, 1969) undoubtedly make a major contribution to these properties. Although minocycline offers improved oral bioavailability over the majority of other tetracyclines, the risk of side effects when used for a protracted period and of the development of resistance has limited it largely to the treatment of acne vulgaris. A recent review suggests that it may offer potential for the systemic treatment of community-associated MRSA and of the significant nosocomial threat of Acinetobacter baumanii (Bishburg and Bishburg, 2009).

Tetracyclines are known to bind strongly to a range of metal ions, with the strongest and most significant binding, in terms of bioavailability, mode of action, mechanisms of resistance, and adverse events, being to magnesium, calcium, iron, and copper (Agwuh and MacGowan, 2006). Chelation of tetracyclines to metal ions in the GI tract adversely affects the absorption of both the tetracycline and the metal ion through the precipitation of insoluble metal-tetracycline complexes (Agwuh and MacGowan, 2006), which provides a scientific rationale for avoiding the administration of tetracyclines alongside metal ion preparations and dairy products. The ability of tetracyclines to coordinate metal ions means that these antibiotics should not be administered to children, due to their ability to sequester calcium and other metal ions at a crucial time in bone and teeth development. In the USA and Australia, children under the age of eight are contraindicated, while in the UK tetracyclines should not be given to children under the age of 12. European guidelines on the third-generation tetracycline tigecycline recommend that it is not used for children and adolescents under the age of 18 years, due to lack of data on its safety and efficacy in these patient groups. The reason for the strong metal ion binding can be seen by considering the tetracycline structure – the lower face of the molecule (as can be seen in Scheme 4.3.9) has several oxygen atoms and so is ideal for binding to a metal ion the C12 (as the enolate) and C11 oxygen atoms are accepted to be the major binding site (Jin et al ., 2007 Palm et al ., 2008).

Scheme 4.3.9 Tetracycline-magnesium ion chelation (Jin et al ., 2007 Palm et al ., 2008)

We will return to tetracycline-magnesium complexes again later in this section, when we consider the uptake of tetracyclines into bacterial cells and also when we look at the mode of action (Subsection 4.3.4) and mechanisms of resistance (Subsection 4.3.5).

In general, the tetracycline antibiotics are not metabolised, except for tetracycline, of which about 5% is metabolised, and tigecycline, of which 5–20% is metabolised (Meagher et al ., 2005) they are excreted by both the urinary (<50%) and the faecal (>40%) routes (Agwuh and MacGowan, 2006). The urinary excretion of tetracyclines has been found to be affected by pH as expected, it is significantly increased at pH values above 8, at which values greater ionisation and hydrophilicity are also to be expected (Jaffe et al ., 1973).

The tetracyclines exhibit a high volume of distribution and generally good tissue penetration alongside their long half-lives and significant post-antibiotic effect, most can be administered once or twice daily (Table 4.3.3).

Table 4.3.3 Pharmacokinetic parameters for selected tetracycline antibiotics (Zhanel et al ., 2004 Agwuh and MacGowan, 2006 Hoffmann et al ., 2007 Barbour et al ., 2010).

We discussed above how metal ion binding adversely affects the absorption of tetracyclines from the GI tract, but it is an important and crucial part of the uptake of tetracyclines into bacterial cells. The chelation to a magnesium ion forms a tetracycline-magnesium cationic complex (with an overall charge of +1), which is transported through the outer membrane into the periplasm by porins, such as the well-characterised OmpF and OmpC examples from Gram negative bacteria (Nikaido, 1994, 2003). You will remember from Section 1.1.2 that porins are protein pores which transport a range of molecules through the outer membrane into the periplasm they are known to be responsible for the transport of other antibacterial agents, such as quinolones and β-lactams, besides tetracyclines (Jaffe et al ., 1982 Mortimer and Piddock, 1993). The mechanism for porin-mediated transport relies upon the Donnan potential 14 across the outer membrane and leads to accumulation of the tetracycline-magnesium cationic complex in the periplasm (Zhanel et al ., 2004). It is probable that the tetracycline-magnesium complex dissociates in the periplasm, perhaps due to the lower pH in this compartment, which is sufficiently acidic to drive the reprotonation of the enol oxygen at C12. The released zwitterionic tetracycline (overall neutral charge) is in equilibrium with a small proportion of the uncharged form, which is weakly lipophilic and able to diffuse through the cytoplasmic (inner) membrane in an energy-requiring process (Scheme 4.3.10) (Nikaido and Thanassi, 1993). Tetracyclines are presumed to adopt a similar uncharged tetracycline diffusion entry route through the simpler cell membrane of Gram positive bacteria.

Scheme 4.3.10 A tetracycline-magnesium complex dissociates to the zwitterion in the periplasm and equilibrates with the neutral form, which enters bacterial cells by passive diffusion (Nikaido and Thanassi, 1993)

You may be wondering why the diffusion of the neutral tetracycline across the bacterial cytoplasmic membrane is energy-requiring. Live bacteria have a difference in pH between the cytoplasm and periplasm of about 1.7 pH units (Nikaido and Thanassi, 1993), so that, after the neutral tetracycline passes through the cytoplasmic membrane, it becomes trapped at the higher pH of the cytoplasm, releasing a proton to form a greater proportion of tetracycline in a more hydrophilic form (overall molecular charge of −1) (Scheme 4.3.11). To maintain the pH of the cell, and the difference with the external pH, the bacteria must continue to transport protons out of the cytoplasm, which is an energy-dependent process (Nikaido and Thanassi, 1993), so it is not actually the diffusion of tetracycline into the cell that is energy-requiring, but the maintenance of pH that is required as a result.

Scheme 4.3.11 In the cytoplasm, the hydrophilic form of the tetracyclines predominates and reforms the tetracycline-magnesium complex (Nikaido and Thanassi, 1993)


The Effect of Kanamycin and Tetracycline on Growth and Photosynthetic Activity of Two Chlorophyte Algae

Antibiotics are routinely used in microalgae culture screening, stock culture maintenance, and genetic transformation. By studying the effect of antibiotics on microalgae growth, we can estimate the least value to inhibit growth of undesired pathogens in algal culture. We studied the effect of kanamycin and tetracycline on the growth and photosynthetic activity of two chlorophyte microalgae, Dictyosphaerium pulchellum i Micractinium pusillum. We measured CFU mL −1 on agar plates, optical density, fluorescence yields, and photosynthetic inhibition. Our results showed a significant effect of kan i tet on the tested microalgae species except tet, which showed a minor effect on M. pusillum. Both antibiotics are believed to interact with the protein synthesis machinery hence, the inhibitory effect of the tested antibiotics was further confirmed by isolation and quantification of the whole cell protein. A significant reduction in protein quantity was observed at concentrations more than 5 mg L −1 , except M. pusillum, which showed only a slight reduction in protein quantity even at the maximum tested concentration of tet (30 mg L −1 ). This study can further aid in aquaculture industry, for the maintenance of the microalgae stock cultures and it can also help the microalgae genetic engineers in the construction of molecular markers.

1. Uvod

Microalgae are gaining importance in medical, pharmaceutical, and food industry. With the increasing applications of microalgae, it is mandatory to investigate growth conditions and potential growth inhibitors. Herbicides, antibiotics, and heavy metals are toxic to microalgae even at low concentrations [1–6]. Studying the survival and adoption of microalgae in the contaminated environment is not an insignificant question and to a certain extent, the microalgae could survive in contaminated environments [7–10].

In the past decade antibiotics use and resistance have been the focus of the world leading organizations, including the Center of Disease Control (CDC) and the World Health Organization (WHO). Alexander Fleming and Howard Walter Florey warned the world first time about the antibiotic resistance while receiving 1945 Nobel Prize for the discovery of penicillin [11]. Antibiotic resistance has been a productive research topic for scientists in the medical field [12]. Anthropogenic activities including use of antibiotics in agriculture, aquaculture, and waste disposal have been linked with the antibiotic resistance [13–15].

Aminoglycosides are the commonly used broad-spectrum antibiotics, that is, streptomycin, kanamycin, and amikacin. Aminoglycosides are characterized as multifunctional hydrophilic carbohydrates with several amino and hydroxyl activities having higher affinities to the prokaryotic rRNA [16, 17]. Suzuki et al. studied the effect of kanamycin on bacterial protein inhibition [18]. Kestell et al. reported the effect of kanamycin and streptomycin on the macromolecular composition of Escherichia coli strains [19]. The inhibitory effect of streptomycin had been reported to microalgae species at a concentration of 0.5 to 150 mg L −1 [20–22]. Galloway reported a halotolerant algae Amphora coffeaeformis resistance to streptomycin [23]. Kvíderová and Henley reported the effect of ampicillin and streptomycin on the growth and photosynthetic activity of halotolerant chlorophyte algae species [24]. However, a limited or no literature is available on the structural studies of aminoglycosides interaction with RNA sequences.

Kanamycin is a broad-spectrum aminoglycoside antibiotic, isolated from bacterium Streptomyces kanamyceticus [25]. It is considered an important medication needed in a basic health system and it has been listed in the WHO’s list of Essential Medicines [26]. Kanamycin interacts with the 30S ribosomal subunit resulting in a significant amount of mistranslation and prevents translocation during protein synthesis [27, 28], whereas tetracyclines bind to the 16S part of the 30S ribosomal subunit and prevent amino-acyl tRNA to attach at A-site of mRNA-ribosome complex, ultimately inhibiting protein synthesis as well as cell growth [29–31].

Kanamycin resistance (Kan R ) is mainly due to the cytoplasmic aminoglycoside phosphotransferase that inactivates kanamycin by covalent phosphorylation. On the other hand, tetracyclines are a group of broad-spectrum antibiotics, but their general application has been shortened because of the inception of antibiotic resistance [32–34]. Cells can become resistant to tetracyclines by one of the three mechanisms: enzymatic inactivation of tetracycline, efflux, and ribosomal protection [35].

Antibiotics tolerance of prokaryotic microorganisms has been described by leading scientists, but there are just a few reports available on the antibiotic tolerance study of eukaryotic microalgae [20, 22, 23, 36]. No doubt, antibiotics are normally considered effective against prokaryotic microorganisms, but they are extensively used in microalgae culture screening [37, 38], in aquaculture, and for screening of genetic transformants [39] hence, there is a need to check the effects of the antibiotics against eukaryotic microalgae.

This work was planned to determine the activity of two important antibiotics, kanamycin sulfate and tetracycline hydrochloride, against the freshwater eukaryotic microalgae species, Dictyosphaerium pulchellum i Micractinium pusillum. Colony forming units, optical density, fluorescence yields, and photosynthetic inhibitions were measured. The antibiotics used in this study are believed to interact with the protein synthesis machinery hence, the whole cell protein was also extracted and quantified.

2. Material and Methods

2.1. Microalgae Cultivation and Treatment

The eukaryotic freshwater microalgae species, Dictyosphaerium pulchellum i Micractinium pusillum, used in this study were obtained from the Korea Marine Microalgae Culture Center (KMMCC), Busan, South Korea. Stock cultures were stored on the modified AF6 agar slants [40]. The cultures were streak plated and purified by subculturing by at least 5-6 times before use. Both microalgae species were cultivated in 250 mL flasks with 150 mL, modified AF6 medium while incubating at

μmol photons m −2 s −1 and 50% humidity. Antibiotics, kanamycin sulfate (Amresco), and tetracycline hydrochloride (Bio101) with different concentrations ranging from 0 to 30 mg L −1 were used. Growth rates were calculated by measuring the absorbance at 750 nm (OD750) on every alternating day [41]. Additionally, all the experiments were repeated three times.

2.2. Screening Tests

The spread plate method according to Markham and Hagmeier [42], with slight modifications, was used to obtain colonies of the tested microalgae on agar plates. 200 μL of the cultured microalgae with approximately adjusted initial cell density (1 × 10 4 cells mL −1 ) was spread plated on AF6-agar plates supplemented with different concentrations of kan i tet ranging from 0 to 30 mg L −1 . Plates were incubated under constant light intensities and the growth was observed for three weeks.

2.3. Modulated Fluorescence and Photosynthetic Inhibition Measurement

Fluorescence yields of algae samples treated with different concentrations of kan i tet were measured by toxy-PAM dual channel yield analyzer (Heinz Walz GmbH, Effeltrich, Germany). The toxicity test is based on extremely sensitive measurement of the effective quantum yield (Y), of photosystem II (PSII), via assessment of chlorophyll fluorescence yield by following the saturation pulse method [43, 44]. Fluorescence of the dark adopted algal samples (

) is measured by using modulated light of low intensity to avoid the reduction of the PSII primary electron acceptor (

) [43]. In order to induce an equilibrium state for the photosynthetic electron transport, prior to measurement of fluorescence, algal cells were adapted to darkness for 20 min.

In the toxy-PAM blue light is used for excitation and fluorescence is assessed at a wavelength above 650 nm. The ( ) fluorescence level corresponds to the fluorescence measured shortly before the application of a saturation pulse. Maximum fluorescence level (

) corresponds to the maximal fluorescence measured during a saturation pulse. The effective PSII overall quantum yield of the photochemical energy conversion was calculated by the formula given by Genty et al. [44].


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