ZA200602769B - Use of tigecycline, alone, or in combination with rifampin to treat osteomyelitis and/or septic arthritis - Google Patents

Description

USE OF TIGECYCLINE, ALONE, OR IN COMBINATION WITH RIFAMPIN TO

TREAT OSTEOMYELITIS AND/C»R SEPTIC ARTHRITIS

T his application claims priority to U.S. Prowisional application 60/500,474, filed on September 5, 2003, which is herein incorporated by reference in its entirety.

FIELD OF THE IN\WENTION “The present invention relates to a novel method of treating osteomyelitis an_d septic arthritis caused by or as a result of bacterial infecti ons. The present invention also relates to treatrment of bacterial infections of the bone, bome marrow, joint, and synovial fl uid.

The pre sent invention further relates to treatment Of antibiotic resistant bacterial infections in theses diseases and tissues.

BACKGROUND OF INVENTION

The last half of the 20" century saw signifTicant progress in the developmert of antibac terial agents. This success fostered the perception that bacterial diseases w~ere more readily cured than any other major disorder, but the emergence of multidrug-resis®ant organisms in the 1990s resulted in serious public Thealth implications. Resistance _has spread to previously susceptible organisms, and s-ome organisms are essentially re=sistant to ail approved antibacterial agents. . Tigecycline, which belongs to the glycylc- ycline class of antibiotics, circurmvents existine g mechanisms of microbial resistance. It demonstrates a broad spectrum o=f antibacterial activity, inhibiting multiple resistan® gram-positive, gram-negative, and anaerobic bacteria. Tigecycline is active against most common pathogens. Tigecycline is active against pathogens such as methicillin-resisstant Staphylococcus aureus (MIRSA), vancoynycin-resistant enterococci (including Enterococcus faecalis), penicillin- resistaant/macrolide-resistant pneumococci, Preveotella spp., peptostreptococcl, mycobacteria, and minocycline-resistant organis ms (Boucher et al., Antimicrob ~dgents

Chem other. 2000; 44(8): 2225-2229, Gales et ad., Antimicrob Agents Chemother—. 2000; 46: 1 9-36, Goldstein et al., Antimicrob Agents Chemother. 2000; 44(10): 2747- 2751).

Tigec ycline is useful in the treatment of respirateory pathogens such as Streprocoeccus pneurnoniae (penicillin sensitive and penicillin resistant), Haemophilus influenzae,

Chlarnydia pneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus (methhicillin-

susceptible and mmethicillin-resistant), aerobic gram-negative rods, and enterococci (vancomycin-sussceptible and vancomycin-resistant ente=rococci). The in vivo results have been very encoumraging and better than would be predict ed based on time above mirimum inhibitory concemntration (MIC) in serum. Tigecycline i=s observed to be a safe antitoacterial agent.

MethicilRin-resistant staphylococci are the most common organisms in infections of the bone and joint (Waldvogel, Infectious Diseases 1988: 1339-1344). The options for treatment of infe=ctions due to these microrganisms are “limited: the sensitivity of clinical strains to quinoBones, clindamycin, cotrimoxazole, and rifampin is variable, and th e sensitivity is oftten limited to glycopeptides, which musst be administered by the pa_renteral route. Resistanece of staphylococci to glycopeptides hans already been described amd represents a ma_jor concern, since those drugs are considered the gold standard for the treatment of ser-ious infections due to methicillin-resistzant staphylococci (Smith, est al.,, N

Engl J Med 1999, 340: 493-501).

Novel dlirugs for the treatment of methicillin-ressistant staphylococcal infections, such as quinupmristin-dalfopristin and linezolid have rescently been introduced in clinical practice (Johns on, et al., Lancet 1999; 354: 2012-201 3, Livermore, J Antimicrob

Chemother 2000; 46: 347-350). However, none haves been fully investigated in clinical studies on the treatment of osteomyelitis.

The tre atment of acute and chronic orthopedic infections is difficult, due &n part to the fact that mzany of the infections result from antibiotic resistant pathogens but also in part due to the location of the infection. Often the therapy requires a prolonged amntibiotic therapy and surgical treatment (Lazzarini ef al., Curr Infect Dis Rep 2002: 4: 4359-445).

Several studies=s have been performed using various arimal model of osteomyeliti_s (Rissing, Infecct Dis Clin North Am 1990; 4: 377-390 ). Despite a prolonged antibiotic treatment, viable bacteria may be still found in the boone. Eradication of more bacteria from the bone: has been associated with a prolonged Curation of antibiotic treatm_ent (Norden, Rev Infect Dis 1988; 10: 103-110). After four weeks of antibiotic treatment, the majority of aratibiotic regimens were unable to eradicate staphylococci from the “bone.

Antibm atic treatment for osteomyelitis is tradi tionally administered by thes intravenous route. However, oral regimens for osteomyelitis have been successfully tested in human tria”1s (Bell, Lancet 1968; 10: 295.297, Femgin et al., Pediatr 1975; 55 :213-223,

Slama ef al., Am J Med 1987; 82 (Suppl 4A): 259-2«61). Unfortunately, the chowice of oral antimicrobials is restricted when dealing with multi-drugs resistant organisms and treatment of theese multi-drug resistant organisms may re=quire the use of parenteral drugs (Tice, Infect DEs Clin North Am 1998; 12: 903-919).

There thus remains a need for a method of treatirg osteomyelitis and/or septic= arthritis caused by bacterial infections, especially those caused by antibiotic resistant bacterial strain s. The present invention fulfills this long -standing need.

SUMMARY OF THE INVE_NTION

The pressent invention provides a method of treating bone or bone marrow infections (often referred to as osteomyelitis) and/or joimt infections and infections o—f'the surrounding tisssues (often referred to as septic arthritis) in a mammal, preferably a h—uman.

The method comprises administering to the mammal a goharmacologically effective amount of tige=cycline and/or an antimicrobial agent seleected from the group consisti ng of rifamycin, rifampin, rifapentine, rifaximin, or streptova_ricin to treat the infection.

Preferably the- antimicrobial is rifampin.

The infection may be caused by a pathogen sele=cted from the group consistirng of gram negatives bacteria, gram positive bacteria, anacrobaic bacteria, and aerobic bactena.

Exemplary ba_cteria include Staphylococcus, Acinetobacter Mycobacterium, Haemogphilus,

Salmonella, Streptococcus, Enterobacteriaceae, Enterococcus, Escherichia,

Pseudomonas, Neisseria, Rickettsia, Pneumococci, Prezvotella, Peptostreptococci,

Bacteroides Legionella, beta-haemolytic streptococci, group B streptococcus and

Spirochetes. Preferably, the infection is comprised of Neisseria, Mycobacterium,

Staphylococc-us and Haemophilus and more preferably- Neisseria meningitidis,

Mycobacterivem tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis,

Streptococcus pyogenes, Streptococcus pneumoniae, Fdaemophilus influenzae, or

Mycobacterizem leprae.

In pre=ferred embodiments the infection is comporised of a pathogen exhibitirg antibiotic res istance. Exemplary antibiotic resistance Mncludes methicillin resistance, glycopeptide resistance, tetracycline resistance, oxytetracycline resistance, doxycycline resistance; chlortetracycline resistance, minocycline resistance, glycylcycline resistance, cephalospori n resistance, ciprofloxacin resistance, nitr-ofurantoin resistance, trimettoprim- sulfa resistarace, piperacillin/tazobactam resistance, meoxifloxacin resistance, vanco mycin resistance, tesicoplanin resistance, penicillin resistance , and macrolide resistance.

A preferred glycopeptide resistance is vancomycin resistance. In another pre ferred embodime=nt, the infection is comprised of S. au reus exhibiting a resistance selected from the group «consisting of glycopeptide resistance, tetracycline resistance, minocycline resistance , methicillin resistance, vancomycin resistance and resistance to glycylcycline antibioticss other than tigecycline.

In another embodiment the infection is comprised of Acinetobacter baumanrii, which ma_y or may not exhibit antibiotic resistance selected from the group consistirg of cephalosporin resistance, ciprofloxacin resistance, nitrofurantoin resistance, trimeth-oprim- sulfa resistance, and piperacillin/tazobactam resistance.

Im another embodiment, the infection is comprised of Mycobacterium absce.ssus that may or may not exhibit moxifloxacin resis tance. In other embodiments the infection is compri- sed of Haemaphilus influenzae, Enter-ococcus faecium, Escherichia coli,

Neisseriam gonorrhoeae, Rickettsia prowazekii, Rickettsia typhi, or Rickettsia ricketr sii.

T he present invention also provides a wse of a pharmacologically effective amount oftigecy cline for treating osteomyelitis and/or septic arthritis in a mammal. In another embodirent, the present invention provides a use of a pharmacologically effective amount of tigecy=cline and an antimicrobial agent selected from the group consisting of rifazmycin, rifampin _, rifapentine, rifaximin, or streptovarixcin to treat osteomyelitis and/or septic arthritis. In another embodiment, the inventio-n provides a use of a pharmacologicaily effectives amount of tigecycline for manufacture of a medicament for treatment of osteomyelitis and/or septic arthritis in a mamrmal. In another embodiment, there is providecd a use of a pharmacologically effective amount of tigecycline and an antimicrobial agent selected from the group ceonsisting of rifamycin, rifampin, rifa-pentine, rifaximi_n, or streptovaricin for manufacture o fa medicament for treatment of osteomyelitis and/or septic arthritis in a mammmal.

BRIEF DESCRIPTIONI OF THE DRAWINGS

“The drawings illustrate certain embodk iments of the present invention and En no way ares meant to limit the scope of the invention.

Figure 1 shows the pharmacokinetics of tigecycline in normal new Zealan.d white rabbits. which establishes serum levels above the minimum inhibitory concentrati on over twelve hours after treatment with 14 mg/kg osf tigecycline.

Figure 2 shows the investigators’ gracling of extent of bone infection as se en in x-

ray images. The data demonstrate the effective treatment of osteommyelitis by tigecyc line and tigecycline in combinatiora with rifampin over controls.

Figure 3 shows the colony-forming units per gram of marreow and bone in each of the treatments, which demonstrates that tigecycline and tigecyclin e in combination with = rifampin were an effective treatment for infection of the bone and infection of the marrow with respect to controls.

Figure 4A provides a graphic depiction of the weights of rabbits throughout the time course of administration «of various antibacterials.

Figure 4B provides a graphic depiction of weight variance=s of rabbits throughout 1 0 the time course of administrat jon of various antibacterials.

Figures 5A and 5B show the peaks and troughs of tigecyc line (14 mg/kg twice daily) and vancomycin (30 mg/kg twice daily) in the serum of infZected rabbits after administration of the respectiwe drugs. The data demonstrate that the antibiotic serum levels were above minimum 1 mhibitory concentrations throughout treatment.

ES

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of treating beone and bone marrow infections in a mammal. Preferably the mammal is human. In a preferred embodiment, the bone or bone marrow infesction causes osteomyelitis. Osteomyelitis is an acute or 20 chronic infection of the bone and/or bone marrow, and includes the related inflammatory process of the bone and its stxructures due to infection with pyogenic organisms. The infection associated with osteomyelitis may be localized or it ma.y spread through the periosteum, cortex, marrow, and cancellous tissue. Common bacterial pathogens causing osteomyelitis vary on the basis of the patient's age and the mechanism of infection. Acute 225 osteomyelitis includes two primary categories: heamatogenous costeomyelitis and direct or contiguous inoculation osteomyelitis.

Heamatogenous osteomyelitis is an infection caused by tacterial seeding from the blood. Acute heamatogenou s osteomyelitis is characterized by &n acute infection of the bone caused by the seeding Of the bacteria within the bone from a remote source. 30 Heamatogenous osteomyelitis occurs primarily in children. The= most common site is the rapidly growing and highly vascular metaphysis of growing bon_es. The apparent slowing or sludging of blood flow as the vessels make sharp angles at thes distal metaphysis predisposes the vessels to thwombosis and the bone itself to locallized necrosis and bacterial seeding. These changes in bone structure may be seen in X-ray images. Acute haematogenous osteomyelitis, despite its name, may have a slow clinical devezlopment and insidious onset.

Direct or contiguous inoculation osteomyelitis is caused by direct con®act of the 5S tissue and bacteria during trauma or surgery. Direct inoculation (contiguous-—focus) osteomyelitis is an infection in the bone secondary to the inoculation of orgarmisms from direct trauma, spread from a contiguous focus of infection, or sepsis after a sLargical procedure. Clinical manifestations of direct inoculation osteomyelitis are mowre localized than those of haematogenous osteomyelitis and tend to involve multiple organisms/pathogens.

Additional categories include chronic osteomyelitis and osteomyelitis secondary to peripheral vascular disease. Chronic osteomyelitis persists or recurs, regardl ess of its initial cause and/or mechanism and despite aggressive intervention. Althougeh listed as an etiology, peripheral vascular disease is actually a predisposing factor rather t han a true cause of infection.

Symptoms of osteomyelitis often include high fever, fatigue, irritabil ity and malaise. Often movement may be restricted in an infected limb or joint. Lo cal edema, erythema, and tenderness generally accompany the infection and warmth may be present around the affected area. Sinus tract drainage may also be present at later st ages of infection. Hematogenous osteomyelitis usually presents with a slow insidio us progression of symptoms, while chronic osteomyelitis may included a non-healing ulcer, sinus tract drainage, chronic fatigue and malaise. Direct osteomyelitis generally preserts with prominent signs and symptoms in a more localized area.

Certain disease states are known to predispose patients to osteomyel itis. These include diabetes mellitus, sickle cell disease, acquired immune deficiency syndrome (AIDS), IV drug abuse, alcoholism, chronic steroid use, immunosuppressio, and chronic joint disease. In addition, the presence of a prosthetic orthopedic device is an independent risk factor as is any recent orthopedic surgery or open fracture.

Several bacterial patho gens are commonly known to cause acute and direct osteomyelitis. For example, acute haematogenous osteomyelitis in newborns (younger than 4 months) is frequently caused by S$. aureus, Enterobacter species, and group A and

B Streptococcus species. In children aged 4 months to 4 years, acute haem_atogenous osteomyelitis is commonly caused by S. aureus, group A Streptococcus species,

Haemophilus influ enzae, and Enterobacter species. I children and adolescents a ged 4 years to adult, acute haematogenous osteomyelitis is commonly caused by S. aureus (80%), group A Stzreptococcus species, Haemophilus Enfluenzae, and Enterobactear species.

In adults, acute hasematogenous osteomyelitis is comnonly caused by S. aureus ard

S occasionally Enterobacter or Streptococcus species. WPrimary treatment has in the= past included a combination of penicillinase-resistant synthetic penicillin and a third-g=eneration cephalosporin. AMternate therapy includes vancomycin or clindamycin and a third- generation cephalosporin. In addition to these above—mentioned antibacterials, ciprofloxacin and rifampin have been used in a combination therapy for adult pataents. In instances where there is evidence of infection with gr=am-negative bacilli, a third- generation cephaleosporin is often administered.

Direct osteomyelitis is commonly caused generally by S. aureus, Enterobacter species, and Pseuedomonas species. Often times direct osteomyelitis is caused by~ a puncture wound thhrough an athletic shoe. In these ca_ses, direct osteomyelitis is commonly caused by S. aure=us and Pseudomonas species. The gorimary antibiotics in this sczenario include ceftazidime or cefepime. Ciprofloxacin is offen used as an alternative tresatment.

In patients with si_ckle cell disease, direct osteomyelitis is commonly caused by S™. aureus and Salmonella species, and the primary choice for treatment is a fluoroquinolonee antibiotic (not in children). A third-generation cephalosporin (e.g., ceftriaxone) i_s an alternative choice=.

For patien_ts with osteomyelitis due to trauma, the infecting agents usually include

S. aureus, coliformn bacilli, and Pseudomonas aerugirosa. Primary antibiotics ar-e nafcillin and ciprofloxacin _. Alternatives include vancomycin and a third-generation cephalosporin with antipseudommonal activity.

Accordingly, as used herein and in the claimss, the term "osteomyelitis" irmcludes haematogenous osteomyelitis, direct or contiguous iraoculation osteomyelitis, chronic osteomyelitis andi osteomyelitis secondary to periphe=ral vascular disease. Osteormyelitis may be the result of infections caused by any of the above described pathogens, but also includes other pasthogens having the ability to infect ®he bone, bone marrow, join_t, or surrounding tissu es.

The term "treating osteomyelitis” includes eraadication of the pathogens/b acteria causing the underlying infection associated with ostesomyelitis, inhibition of bacterial growth, reductiorn in bacterial concentration, reduction in recovery time from infeection,

improvement, elimination, or reduction of symptoms of infection such as swellmng, necrosis, fever, pain, weakness, and or other indicateors as are selected as approgpnate measures by those skilled in the art.

Curresntly, the primary treatment for osteomyelitis is parenteral antibioti_cs that penetrate bone and joint cavities. Treatment is requ_ired for at least four to six voveeks.

After intravenous antibiotics are initiated on an inpatient basis, therapy may be continued with intravemnous or oral antibiotics, depending on tie type and location of the infection, on an outpatient basis.

For eexample, osteomyelitis caused by S. aur—eus infection is generally treated with 2 grams of c=loxacillin administered intravenously oT parenterally every six hou._rs for at least the init-ial 14 days or for the entire treatment course of up to six weeks. O=ther treatments a re cefazolin administered at 1 to 2 grams every eight hours for six ~weeks or 600 mg of clindamycin every eight hours for six weeks.

Oste omyelitis caused by beta-haemolytic streptococci is generally treatwed intravenousBy or parenterally with benzylpenicillin at two million IU every fou- to six hours for tw-o to four weeks. Infections by Salmonella spp. are treated with cipmrofloxacin at 750 mg omrally every 12 hours for six weeks.

Trea tment of osteomyelitis caused by Haenmophilus influenzae in childr—en is generally with intravenous or parenteral administration of 25-50 mg cloxacillira every four to six hours for four to six days plus ceftriaxone at =50-75 mg/kg every 24 hourss for four to six days. This treatment is followed by amoxicillin at 15 mg/kg plus oral clavialanic acid (maximum 500mg) every eight hours for four week—s.

In neonates, treatment is accomplished with_ intravenous or parenteral ¢ loxacillin at 25-50 mg/kg every four to six hours for four to six ~days plus intravenous or pa=xenteral cefotaxime zat 50-75 mg/kg every eight hours for fo ur to six days. Treatment iss followed by amoxicil lin at 15 mg/kg plus oral clavulinic acid! (maximum 500mg) every eight hours for four weeks.

Infection in children with S. aureus is gener-ally treated with intravenoums or parenteral aedministration of 25-50 mg cloxacillin e~very four to six hours for fo- ur to six days plus ceftriaxone at 50-75 mg/kg every 24 hounrs for four to six days. This treatment is followed by~ cloxacillin at 12.5 mg/kg orally every ssix hours for three to four weeks.

Treastment of infection in children with Salmonella spp. depends upon t=he susceptibility of the pathogen. Treatment choices imclude cloxacillin plus ceftriaxone followed by either sufamethoxazole at 20 mg/kg and trime=thoprim at 4 mg/kg orally every 12 hours for si» weeks, or amoxicillin at 7.5-15 mg/lkg orally every 12 hours for six weeks, or ciprofloxacin at 10-15 mg/kg every four to six h_ours for four to six days pluss cefotaxime at 50-75 mmg/kg intravenously every eight hour-s for four to six days, follow—ed by sulfamethoxazole and trimethoprim or amoxicillin or c-iproflaxacin.

Another embeodiment of the present invention prov=ides methods of treating joirt infections and/or surrounding tissue infections in a mammal. Preferably the mammal is human. In a preferrexd embodiment, the joint infection ancl/or surrounding tissue infec tion causes septic arthriti s.

Septic arthritis is an infection of the joint and surreounding tissues and results isn joint inflammation c=aused by the presence of live intra-articular micro-organisms. Septic arthritis most comm only occurs secondary to osteomyelit 1s, especially in childhood, a_nd arises as a result of bacterial infection.

Infection of the joint can occur by several routes. Most commonly, the spreach of the infecting pathogen is haematogenous. Frequently sepwtic arthritis arises from infections or abscesses in the sskin. Sepsis in the mouth and teeth or— after dental procedures or ira association with infeection of the respiratory or urogenital tract can also lead to septic arthritis. Direct permetrating trauma to the joint with shargp objects or from major traurmatic injury can lead to joint infection as well. Joint aspiration. or injection and surgical procedures such as joint replacement may also result in jeoint infection. Additionally, osteomyelitis often spreads to involve the joint. This is especially common in young children. Finally, infection of the soft tissues adjacent tos the joint, such as inflamed boursae " ortendon sheaths, can spread to involve the joint space. Spread of infection by the haematogenous route is still the most frequent cause of jwoint sepsis.

Symptoms of septic arthritis include malaise and fever, acute hot joint or join_ts together with acute inflammation: swelling and joint effi_sion, redness, pain and loss of function.

The most coommon causative organism of septic aarthritis is Staphylococcus arureus.

In neonatal septic arthritis, Escherichia coli and Haemophilus influenzae are also cormmon pathogens. In chilciren up to 5 years Haemophilus influenzae is the most common cause of haematogenous joi nt sepsis. Gram-negative intestinal b=acteria are also common pathogens in the elderly and t hose with diabetes mellitus or prosthetic joints. In cases of penetmrating injury, and in intra-venous drug abusers, infection with Pseudomonas aeruginosa or

Staphylococcus epidermidis are often found. In healthy young adults NAeisseria gonorrhoeae or meningococcal infection are sometimes the cause of se=ptic arthritis.

Chronic low grade septic arthritis, especially in the spine, can be the re=sult of infection with micro-organisms such as Mycobacteria or Brucella abortus. Furthermore, within acquired immune deficiency syndrome sufferers the range of joint pathaogens is diverse.

Some of the most cornmon septic arthritis pathogens include, bwt are not limited to, (1) Gram positive: Staphylococcus aureus (80% cases), Streptococcus pyogenes/pneumoniae; (2) Gram negative: Haemophilus influenzae, NJeisseria gonorrhoeae/meningitidis, Pseudomonas aeruginosa, Bacteroides fragilis, Brucella species, Salmonella species, fusiform bacteria; (3) acid-fast bacilli: Mycobacterium tuberculosis, atypical mycobacteria; and (4) Spirochaetes: Leptospira icterohaemorrhagica.

Accordingly, the terrn "septic arthritis” as used herein and in time claims includes infections of the joint and suarrounding tissues caused by the above list ed pathogens as well as any other pathogens having the ability to infect the joint and surrounding tissues.

Surrounding tissues include, but are not limited to, surrounding muscl -, related tendons, connecting bones, bursae, tendon sheaths, synovium, synovial fluid, amd related cartilage.

The term "treating septic arthritis” includes eradication of the pathogens/bacteria causing the underlying infection associated with septic arthritis, inhibmtion of bacterial growth, reduction in bacteri al concentration, reduction in recovery tine from infection, improvement, elimination, or reduction of symptoms of infection suck as swelling, necrosis, fever, pain, weakness, and or other indicators as are selected. as appropriate measures by those skilled ira the art.

Septic joints are usally treated for four to six weeks while infSected arthroplasties are treated for four to six weeks or more. (Calhoun et al., Am. J. of Szurgery 1989; 157: 443-449, Calhoun et al., Archives of Otolaryngology - Head and Nec Surgery 1988; 114: 1157-1162, Gordon et al., Antimicrob Agents Chemother 2000; 4410). 2747-2751,

Mader et al., West J Med 1988; 148: (5)568, Mader et al., Orthopaedic Review 1989; 18: 581-585, Mader et al. Drugrs & Aging 2000; 16(1): 67-80). These lemgthy antibiotic treatments become even more problematic when drug resistant bacteria, such as methicillin-resistant Staphylococcus aureus, is present.

Prior to characterization of the pathogen, treatment of septic arthritis in adults usually begins with 2 gm cJoxacillin given intravenously or intramuscularly every six hours in combination with 1-2 gm ceftriaxone every 24 hours. In childre n over two months, treatment includes cloxaci1lin intravenously or intramuscularly at 25-50 mg/kg up to a maximum of 2 gm every six hours in combination with ceftriaxone 225-50 mg/kg up to a maximum of 2 gm every 24 hours. In neonates, treatment includes clomacillin intravenously or intramuscularly at 50-75 mg/kg up to a maximum of 2 g=m intravenously every eight hours. Other antibiotic treatments include cefotaxime, fluclo-xacillin, benzyl and penicillin.

Once the pathogen has been identified, the common course of treatment is based on the infecting pathogens present. For example, when it is determined t hat the infection comprises Staphylococcus aureus, septic arthritis is often treated with cloxacillin intravenously every six hours, or cefazolin every eight hours, or clindamycin every eight hours, the chosen treatment lasting for two to three weeks. Methicillin-resistant S. aureus is treated with parenterally administered vancomycin.

Antibiotic treatment of oste omyelitis and septic arthritis is still a <hallenge for the physician, Many orthopedic infect ions are acquired in the nosocomial eravironment (Holtom et al., Clin Orthop 2002; £403: 38-44). Further, the causative agsents of such infections are often multi-drug resistant. Staphylococci are the most common nosocomial and drug resistant organisms, but gram negative pathogens may be involwed as well (Cunha, Clin Infect Dis 2002; 35: 287-293).

Infections due to methicillir-resistant Staphylococcus aureus, cormpared with those due to methicillin-susceptible S. ate reus, are more difficult to treat and many have a poorer prognosis (Cosgrove et al., Clin Infect Dis 2003; 36: 53-59). Therapeuti_c options for these infections are limited. The only drugs with a constant efficacy against all the staphylococcal strains, and which heave been extensively studied in the tresatment of bone infections, are glycopeptides. Unfortunately, resistance to these antibioti cs has been already recognized as a major prob lem in the treatment of gram positive gpathogens.

Enterococci resistant to vancomycin are diffused worldwide and such a ressistance has been demonstrated as potentially transmittable to other gram positive org anisms in vitro (Noble, er al., FEMS Microbiology Letters 1992; 72:195-198). Moreovem, sporadic strains of vancomycin-resistant Staphylococcus aureus have been isolated in several countries (Hiramatsu, Am J Med 1998; 104:7 S-10S, Hamilton-Miller, Infection 2002; 30: 118-124).

Therefore, the availability of alternative antimicrobial agents for the treat—-ment of multi- drug resistant pathogens is of paramount importance.

]

Tigecycline (formerly and often still referred to as "GARR-936") 1s a 9-- butylglycylamido synthetic derivative of a new class of antibiot ics called glycylcyclines.

This new class of tetracycline clerivatives has demonstrated exc ellent in vitro activity against a large number of gram positive and gram negative, aereobic and anaerobic organisms, including methicillan-resistant Staphylococcus auremis (MRSA), vancomycin- resistant enterococci (including Enterococcus faecalis), penicilBin-resistant/macrolide- resistant pneumococci, Prevoteella spp., peptostreptococci, and _Mycobacterium spp. (Boucher et al., Antimicrob Agzents Chemother. 2000; 44(8): 22225-2229, Gales et al.,

Antimicrob Agents Chemother—. 2000; 46: 19-36, Goldstein et «al, Antimicrob Agents

Chemother. 2000; 44(10): 2737-2751). Tetracyclines are bacteriostatic agents, which act to inhibit bacterial protein synthesis. The glycylcyclines have Wbeen developed to overcome the bacterial mechamisms of resistance to tetracyclin=-es, even though their exact mechanism of action has not yet been determined (Rasmussen et al., Antimicrob Agents

Chemother 1995; 38: 1658-16560).

Tigecycline concentrates in bone, bone marrow, joint, Zand synovial fluid as well as many other organs and tissuess of interest. Furthermore, it has “been discovered that tigecycline concentrates in infected portions of the above described tissues. Studies of the pharmacokinetics of intravenous tigecycline in humans have shhown that there is a rapid distribution phase, with a proRonged half-life (40 to 60 hours) zand a high volume of distribution at steady state (7 to 14 L/kg). Animal studies with radiolabeled tigecycline suggest that this rapid distribuation phase and high volume distmribution at steady state represent penetration of tigecZycline into tissues including lung- and bone.

For example, the distribution of tigecycline in rat tissues has been shown in

Sprague-Dawley rats when given ['4C] tigecycline at a dosage- of 3 mg/kg by 30-minute

IV infusion. In general, radioactivity was well distributed to rmost tissues, with the highest overall exposure observed in bone. Exposure in tissues showi ng the highest concentrations were as follows: bone>bone marrow>salivary gland, thyroid, spleen, and kidney. In each of these tissues, the ratio of area under the comncentration-time curve (AUC) in tissue to AUC in plasma was greater than 10. In thi s study, the ratio of AUC in the rat lung to AUC in the plasma was 4.4. Additionally, it haas been demonstrated that intravenously administered ti gecycline penetrates bone tissue in humans and intravenous administration extends concentration of tigecycline in synovial fluid in human over time.

The inventors have d@scovered that tigecycline is a useful treatment of osteomyelitis and septic arthmritis. The antimicrobial spectrum is broad, including all the pathogens found in nosocom ial bone and joint infections. The p-harmacokinetic properties are favorable, since the drug may be administered twice daily. Moreover, bone penetration and drug levels a_bove the minimum inhibitory concentration (MIC) were found in almost every samples collected. Minimum inhibitory comncentration is a method of determining the efficacy of a- compound in inhibiting bacterial growth. It is the lowest concentration of an antimicrobial agent that inhibits growth of a micro-organism and should correspond to concentrations required in sera of the mam_mal for the most minimal treatment. Additionally, tige=cycline provided a good safety profile in humans, demonstrating that the antimicrobial should be suitable for cliniczal studies on orthopedic infections.

Accordingly, one asp ect of the invention provides a method for treating infections of the bone, bone marrow, joeint and surrounding tissue, and a meethod for treating osteomyelitis and/or septic amrthritis in a mammal by administering to the mammal a pharmacologically effective amount of tigecycline. The bone, b=one marrow, joint and surrounding tissue infections. and osteomyelitis and/or septic artknritis and maybe caused by any of the commonly found pathogens, such as the pathogens di=scussed above, which include gram negative bacteria, gram positive bacteria, anaerobic bacteria and aerobic bacteria. For example, the irmfection may be comprised of, but neot limited to,

Staphylococcus, Acinetobact<er, Mycobacterium, Haemophilus, Salmonella, Streptococcus,

Enterobacteriaceae, Enteroc occus, Escherichia, Pseudomonas, Neisseria, Rickettsia,

Pneumococci, Prevotella, Pe-ptostreptococci, Bacteroides Legiomella, beta-haemolytic streptococci, and group B str-eptococcus. In preferred embodiments, the infection is comprised of Neisseria, Myceobacterium, Staphylococcus, and Haaemophilus. In more preferred embodiments the irfection is comprised of Escherichicz coli, Neisseria meningitidis, Neisseria gonoarhoeae, Mycobacterium tuberculos=is, Staphylococcus aureus, Staphylococcus epidezrmidis, Streptococcus pyogenes, St. reptococcus pneumoniae,

Haemophilus influenzae, Ent erococcus faecium, Rickettsia prow~azekii, Rickettsia typhi,

Rickettsia rickettsii, Mycobacterium leprae, Mcyobacterium absecessus, or Mycoplasma pneumoniae.

In one embodiment o=f the present invention there is provided a method of treating infections of the bone, bone rmarrow, joint and surrounding tissue, and a method for treating osteomyelitis and/or septic arthritis caus<ed by the bacterial strains (such ass those describezd above) that demonstrate antibiotic-resi stance by administering a pharmaceutically effective amount of tigecycline. For example, the exhibited resi=stance may be,. but is not limited to, methicillin resistan <e, glycopeptide resistance, tetrac- ycline

S resistan-ce, oxytetracycline resistance, doxycyclime resistance; chlortetracycline resistance, minocyscline resistance, glycylcycline resistance, cephalosporin resistance, ciprofleoxacin resistan ce, nitrofurantoin resistance, trimethoprirn-sulfa resistance, piperacillin/taz=cbactam resistan ce, moxifloxacin, vancomycin resistance , teicoplanin resistance, penicillin resistan ce, and macrolide resistance.

In a preferred embodiment, the glycopeptide resistance is vancomycin resi stance.

In another preferred embodiment, the infection is comprised of S. aureus exhibitirg resistan_ce from either glycopeptide resistance, testracycline resistance, minocycline resistance, methicillin resistance, vancomycin resistance or resistance to a glycylc ycline antibiotic other than tigecycline.

In another preferred embodiment, the inf ection comprises Acinetobacter baumarnii that may or may not exhibit antibioti« resistance such as cephalosporirm resistarmce, ciprofloxacin resistance, nitrofurantodin resistance, trimethoprim-sulfa resistanmce, and piperacillin/tazobactam resistanc €. In another embodiment, the infection is comprised of Mycobacterium abscessus that ma-y or may not exhibit moxifloxacira resistammce.

In treatment of humans and other mamm als, tigecycline is most commons administered intravenously, although other admi nistration paths are available to ome of skill in the art. Doses of up to 100 mg administered during a one-hour infusion can be tolerate=d in human subjects. Twice-daily admin istrations over nine days of 75 mg or more in 200 el infusions over one hour to subjects having been fed 30 minutes before 1 nfusion resulted in gastrointestinal intolerance in all subjects including nausea and vomitimng.

Twice-alaily administration of 25-50 mg in 200 rl infusions over one hour was to lerated.

A singl e infusion of 100 mg was also tolerated resulting in mean peak serum concentrations of 0.9 to 1.1 micrograms/ml.

Administration of 14 mg/kg twice daily to New Zealand White Rabbits resulted in steady Mevels higher than the minimum inhibitor—y concentration. See Figure 1. The minimum inhibitory concentrations (MIC) and rminimum bactericidal concentrations (MBC) for tigecycline for the MRSA strain usecl in this study were less than 0.2 mg/ml and k 0.2 pg/ml, respectively. Measmuring the MBC provides a method -of determining the efficacy of a compound in killing bacteria. The MBC technique establishes the lowest level of a bactericidal agent that will kill at least 99.9% of the orgzanisms in a standard inoculum.

MIC and MBC were determined by Mercier et al. for tigecycline against vancomycin resistant E. faecium to be 0.125 ug/ml and between 16 and 32 pg/ml, respectively. For S. aureus, minimum inhibitory concentrations and minimum bactericidal concentrations were between 0.25 and 1 pg/ml and 16 and 64 ug/ml, respectively. In _a compassionate use study, the inventors found the mminimum inhibitory concentration of tigecycline against M. abcessus in a human patient to be 0.25 pg/ml.

In mammals, methicillin-resistant S. aureus may be treatecd with tigecycline in the range of 5 mg/kg to 60 mg/kg ®wice daily, more preferably 10 mgz/kg to 40 mg/kg, more preferably 12 mg/kg to 20 mg/Bkg. Appropriate dosages for treatnent of other pathogens will be apparent to one of skill in the art.

In a compassionate use study, one human patient suffered from spina bifada with resultant paraplegia. The patient was severely allergic to sulfa drags and presented with methicillin-resistant bacteremiza from infected heel decubitis. The= patient also had skin breakdown over the right ischitam. The ulcer was debrided, but it did not heal. An MRI revealed osteomyelitis and a se ction of the bone was positive for infection from

Acinetobacter baumannii.

The A. baumanii was resistant to cephalosporins, ciproflox=acin, nitrofurantoin, and demonstrated intermediate resistance to trimethoprim-sulfa and pi—peracillin/tazobactam.

The organism was susceptible to imipenem, gentamicin, and tobra_mycin. The patient was treated with meropenem and tobramycin. Meropenem was later replaced with aztreonam due to eosinophilia. Aztreonan was later discontinued because of persistent eosinophilia.

Tobramycin was also discontinmied because of increased creatinine=. The patient was then treated with tigecycline for two months with either 50 mg every 12 hours or 50 mg every 24 hours. Within one month of receiving treatment with tigecyclire, an MRI showed resolution of the osteomyelitis zand marked improvement was seen in fluid collected from right ischial area. The patient was reported doing well ten weeks [ost treatment with tigecycline.

In another compassionate use study, a patient with anhydro tic ectodermal dysplasia with immunodeficiency had a tlaree and one-half year history of ve=rtebral osteomyelitis with a Mycobacterium abscessus infection . Debridement was accomplished afer one year of infection with placement of hardware. “The patient showed some improvement with cefoxitan, clarithromycin, and amikacin. The amikacin was later stopped due to renal damage. Linezolid and azithromycin were later added to the treatment regime=n. The organism was determined to be resistant to moxifloxacin.

The patient presented later with a new vertebral osteomyelitis just abo ve the site of the old infection. A biopsy was performed and it was determined that no addmtional debridement was needed. The organism was found to be sensitive only to cefoxitin. It was determined that an additional antimicxrobial agent would be helpful and tthe organism was found to be susceptible to tigecycline - Tigecycline was administered up —to MIC 0.25 micrograms/ml. The patient’s white blood cell count was normal while hypogammaglobulinemia was present andl lymphocytic function decreased. "Whe patient had also been under treatment with 1L-12, but IL-12 was held during antibiotic treatment.

The patient was reported to be doing well a year after the treatment.

Another embodiment of the present invention provides a method of tr-eating infections of the bone, bone marrow, joint and surrounding tissue, and a metlod for treating osteomyelitis and/or septic arthritis in a mammal, preferably a humam, comprising administering to the mammal a pharmaco logically effective amount of tigecy-cline and an antimicrobial agent from the ansamycin family, which includes the rifamycira and the streptovaricin groups of antibiotics. The xifamycin family includes rifampin, rifapentine, rifaximin, and preferably, rifampin. These macrocyclic antibiotics have bact ericidal activity because of their propensity for binding to RNA polymerase. These amntibiotics are useful in combination with tigecycline because they effect different steps in boacterial protein synthesis. While the rifamycins effect the activity of RNA polymera se and limit production of messenger RNA, tigecycline effects the activity of ribosomes =and the production of proteins from the messenger RNA. The mode of action of tige=cycline appears to be related to inactivation of the 70S ribosomes through binding to- a tetracycline-binding site in the 30S ribosomal subunit with a somewhat diffe rent orientation than does tetracycline. (Bauers et al., J. Antimicrob Chemother. 22004; 53(4): 592-599).

The present inventors have discovered that tigecycline in combinatio- n with an antibiotic of the rifamycin class of antimicrobials provides additive antimicreobial effect in infected tissue. In an investigation with rabbits inoculated at the tibia with nmethicillin-

resistant S. aureus, treat ment of osteomyelitis with tigecycline in combination with rifampin demonstrated ro infection in bone in 10 rabbits whaile controls showed infection in 11 of 15 rabbits. Treztment in bone marrow also demons trated no infection in 10 rabbits while controls skhowed 5 infected rabbits of 15 rabbits tested. Furthermore, treatment of osteomyelitis in rabbits with tigecycline alone Clemonstrated infection in the bone of one rabbit of 10» and no infection in the marrow.

In mammals, rifampin treatment may be in the range= of 10 mg/kg to 100 mg/kg twice daily, more prefer able it may be in the range of 20 mg/kg to 70 mg/kg twice daily, more preferably it may be in the range of 30 mg/kg to 50 mg/kg twice daily. In New

Zealand White rabbits irfected with MRSA, treatment of 40 mg/kg resulted in bactericidal activity. The minimum inhibitory concentration and minimum bactericidal concentration levels for rifampin agairst the MRSA strain were 0.78 ug/m land 1.56 pg/ml, respectively, yielding a ratio of 0.5.

Human oral administration of rifampin is available w~ith capsules of 150 and 300 mg. Following a single 600 mg dose in healthy human adults, peak serum concentrations averaged 7 micrograms/mml but with wide variance from 4 to 32 micrograms/ml.

Administration of 600 mag intravenously to healthy human aciults over 30 minutes resulted in mean peak serum concentrations of about 17 micrograms/ml.

Administration o ftigecycline is preferably administered intravenously or intramuscularly, while ri fampin may be administered intrave=nously, intramuscularly, orally or by other means of administration known to the art s uch as transbuccal, intrapulmonary or transdermal delivery systems. Co-adminisstration may include a combination of any of th ese methods. For example, tigecycl-ine may be administered intravenously while rifarmpin may be administered orally. Ced-administration includes simultaneous or sequenti al administration, in any order and dlloes not necessarily imply administration at the sare time or same day or same time comurse schedule. Preferably, concentrations of both tiggecycline and rifampin are concurrertly maintained well above the minimum inhibitory czoncentration.

In a trial by the irmventors, a group of rabbits infected —with methicillin-resistant S. aureus and treated with tigecycline showed lower colony forrming units in bone and marrow than the infected , untreated control group or the group treated with vancomycin at the end of the treatment period. The MIC and MBC for tigec ycline (0.2 pg/ml) were lower than that of vancomycin (0.39 ig/ml and 0.78 pg/ml), which is more conducive to the resolution of osteomyelitic infections. The association of tig ecycline and rifampin allowed the complete eradicatieon of bacteria from the bone and rnarrow, whereas in the vancomycin plus rifampin group a sample was still positive. Seez Figure 3. Treatment was successful with subcutaneous administration of 14 mg/kg of tige=cycline twice daily and oral administration of 40 mg/k g of rifampin twice daily. These «data demonstrate that osteomyelitis in rabbits with maethicillin-resistant S. aureus infection is effectively treated with a combination of tigecycl ine and rifampin.

Accordingly, given the disclosure presented herein, suche as the dose and treatment regimens (i.e. length and mode of administration, and time coursse of therapy) used in the above described compassionat e use studies, typical dose and tre atment regimens of common antibiotics administemred to patients to treat infections vavith the listed pathogens, and dose and treatment regimens used in the rabbit study, one skilled in the art would appreciate the appropriate dosee and treatment regimen to admin ister to a mammal to achicve a pharmacologically e=ffective amount of tigecycline ancd/or additional antimicrobrials such as rifampein, to treat osteomyelitis and/or septic arthritis. One skilled in the art would appreciate tha t factors such as the extent of the infection, overall health, weight, and age of the patient would effect the desired dose and_ treatment regiment.

The term "pharmacolo gically effective amount” means, consistent with considerations known in the amt, the amount of antimicrobial ag ent effective to achieve a pharmacologic effect or therapeutic improvement without undu e adverse side effects, including but not limited to, irhibition of bacterial growth, reduction in bacterial concentration, reduction in recovery time from infection, impro vement, elimination, or reduction of symptoms of infection or other disease such as sweslling, necrosis, fever, pain, weakness, and or other indica®ors as are selected as appropriate measures by those skilled intheart.

Another embodiment of the present invention provides &he use of tigecycline with or without an antimicrobial agent selected from the group consi sting of rifamycin, rifampin, rifapentine, rifaximi_n, or streptovaricin (preferably rifampin) for the manufacture of a medicament for treatment infections of the bo ne, bone marrow, joint and surrounding tissue, and osteormyelitis and/or septic arthritis in a_ mammal, preferably a human.

Another embodiment gprovides a pharmaceutical composition for the treatment of infections of the bone, bone marrow, joint and surrounding tissmue, and osteomyelitis and/or septic arthritis in a mammal, preferably a human, comprisimmg tigecycline, with or without an antimicrobial agert selected from the group consisting «of rifamycin, rifampin, rifapentine, rifaximin, or stregptovaricin (preferably rifampin), and pharmaceutically acceptable diluents, preservakives, solubilizers, emulsifiers, adjuva_nts and/or carriers conventionally used in pharmaceutical and veterinary formulations. The present pharmaceutical formulations may be adapted for administration to humans and/or animals.

Another embodiment of the present invention provides the use of tigecycline with or without an antimicrobial a gent selected from the group consistirag of rifamycin, rifampin, rifapentine, rifaxim in, or streptovaricin (preferably rifamepin) for manufacture of a medicament for treatment of infections of the bone, bone marrow, joint and surrounding tissue, and osteomyelitis and/or septic arthritis in a mammal, preferably a human. : It is to be understood -that in the various embodiments of th e present invention, tigecycline and/or rifampin ox other antimicrobials may by present as pharmaceutically acceptable salts thereof. For «example, such salts may include but are not limited to the hydrochloride, sulfate or phosphate salts. They may also include thhe acetate, citrate or lactate salts, for example.

The medicament or pharmaceutical composition is adminis tered at a dose to achieve a pharmacologically effective amount of the tigecycline amd a pharmacologically effective amount of an antimi crobial agent selected from the group consisting of rifamycin, rifampin, rifapentimne, rifaximin, or streptovaricin (prefemrably rifampin). The pharmaceutical composition zand/or medicament further comprise ppharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvamts and/or carriers.

These may include but are not limited to, saccharose, mannitol, sorbitol, lecithins, polyvinylpyrrolidones, microcrystalline celluloses, methylcellulosess, carboxymethylcelluloses, hyd roxyethylcelluloses, hydroxypropyl celluloses; starches, polyacrylates, ethylcelluloses., hydroxypropyl cellulose, hydroxyprepylmethylcellulose and their derivations, triacetirm, dibutylphthalate, dibutylsebacate, cE tric acid esters, polyethyleneglycols, polypropyleneglycols, polyvinylpyrrolidone, Ractose, sucrose, magnesium stearate, talc, or sxlicone oil.

For oral administratior, the pharmaceutical formulations may be utilized as, for example, tablets, capsules, emulsions, solutions, syrups or suspensi ons. For parenteral administration, the formulatioms may be utilized as ampoules, or otherwise as suspensions, solutions or emulsions in aqueous or oily vehicles. The need for su spending, stabilizing and/or dispersing agents will of course take into acco-unt the solubility of the active compounds in the vehicles which are used in particul ar embodiments. The formumlations may additionally co ntain physiologically compatible preservatives and antioxidarts.

The pharmacceutical formulations may also be= utilized as suppositories wi th conventional suppossitory bases such as cocoa butter sor other glycerides. Alternastively, the formulations may bee made available in a depot form that will release the active composition slowly in the body, over a pre-selected time period.

The followirnag examples are given for the pur—pose of illustrating various embodiments of the= invention and are not meant to 1i mit the present invention in any fashion.

EXAMPLES

EXAMPLE 1: Tre=atment of osteomyelitis in rabbits with tigecycline

This exampMe shows the treatment of osteomwyelitis in rabbits with tigecyc=line and tigecycline in comb ination with rifampin. Comparis=on studies with vancomycin and the combination of vanecomycin with rifampin were also performed. Data demonstrate improved antimicrowbial efficacy with tigecycline over vancomycin, and with tige=cycline in combination with rifampin over vancomycin in combination with rifampin. Add_itionally, tigecycline in comb-ination with rifampin provided complete protection against methicillin- resistant S. qureus vavithin its test group.

Generation of Stanclard Curves for Diffusion Bioasszays

Normal NZ™W rabbit serum (Fisher Scientific) and normal, uninfected rab bit tibia bone were used to generate standard curves for tigec—ycline (Wyeth-Ayerst Resea-xch, Pearl

River, New York), “vancomycin (Abbott Laboratoriess, Chicago, Illinois), and rifa_mpin (Merrell Pharmaceuaticals Inc. Kansas, Missouri). Bi oassays were performed wit_h each drug to generate thes standard curves for antibiotic co=ncentration in serum and/or tibial bone.

The organism used for the bioassay was Bacillus cereus ATCC11778. Serum standards were prepared using two-fold serial dilutio-ns with either antibiotic to yield concentrations of 225 pg/ml to 0.20 pg/ml drug in No rmal NZW rabbit serum. Bone eluate standards were prepared for tigecycline by thoroughR y cleaning noninfected rabbit tibias with 70% ethanol ir a sterilized fume hood. Each titoia was broken into small chops of approximately 0.3 cm’ using a grinder. The chips were placed into a sterile, 50 mi conical centrifuge tube ard weighed. One milliliter of sterile, 0.9% normal saline was add ed for each gram of bon € chips. The solution was thoroughly vortexed for two minutes. The resulting bone elvaate was allowed to shake at 180 rpm_ in a cold room at 4°C, for 1 2 hours.

S The samples were centrifuged at 4000 rpm for 3 minutes prior to assay, to pellet tie chips.

The diameeter of the zone of growth inhibition around each well was measumred, in millimeters. A standard curve was generated for tigecycline concentration in both: serum and bone eluate aand for vancomycin in serum by plotting the known antibiotic concentration against its resulting zone of inhibition measurement. :

Pharmacokinetic s of Tigecycline

A baseline group of 6 uninfected rabbits were subcutaneously administeread 14 mg/kg tigecyclinae, reconstituted in sterile water, every 12 hours, for a period of 8 days.

Blood samples were drawn at the following approximate intervals, post-initial ant_ibiotic treatment: 1 hou, 3 hours, 6 hours, 12 hours, 171 houars and 180 hours (time of samcrifice).

One-half millilit=er of blood was collected with standard techniques. Samples were immediately placed into sterile, 1.5 mi centrifuge tubes. Following euthanasia, booth tibias were thoroughly cleansed with 70% ethanol and then harvested, after removal of =all soft tissue. Tibias w-ere placed into separate, sterile S50 mB centrifuge tubes and stored at -70°C.

Serum samples were stored at -70°C until the bioassay was performed. Beone samples were pr-epared as previously described. Seecled agar plates were preparesd and samples were lo aded in triplicate, to the seeded platess and incubated at 30°C for M8 hours.

The diameter of the zone of growth inhibition around each well was measured an d concentrations of tigecycline were extrapolated from the standard curve.

Minimum Inhib_itory Concentration and Minimum Bactericidal Concentration

Determinations

The min_imum inhibitory concentrations (MIC) of tigecycline, vancomyci n and rifampin were dietermined using an antibiotic two-fold tube-dilution method. Thee minimum bactericidal concentrations were also determined. The limits of sensitivity of this method werre 25 pg/ml to 0.20 pg/ml.

Induction of Tibial Osteomyeli tis

A localized S. aureus o steomyelitis was percutaneously indiaced in the left lateral tibial metaphysis of all rabbits within all six study groups. The strazin of methicillin- resistant S. aureus was obtaine d from a patient with osteomyelitis u ndergoing treatment.

Preparation of the Infeactive Media: S. aureus was incubatecd overnight in Mueller

Hinton Broth(Difco Laboratormes, Detroit, Michigan) medium spike=d with 40 pg/ml oxacillin, at 37°C. The bacteria! concentration of the culture was ad justed to 107

CFU’s/ml.

Rabbit Infection Procecdure: New Zealand white rabbits (Ray Nicholl's Rabbitry,

M0 Lumberton, Texas), eight to 12 weeks old and weighing 2.0 to 3.5 k=g, were utilized for the study. After anesthesia was gizven, an 18-gauge needle was inserted percutaneously through the lateral aspect of th-e left tibial metaphysis into the intrarmedullary cavity. Next, 0.15 ml of 5% sodium morrhuzate (American Regent Laboratories, I nc., Shirley, New

York), 0.1 ml of S. aureus (10~ CFU/ml), and 0.2 m] of sterile, norrmal saline, 0.9%, were

BS injected sequentially. The infesction was allowed to progress for 2 vveeks, at which time the severity of osteomyelitis w—as determined radiographically (TabMe I).

Treatment Groups: At the end of two weeks, post infection, the rabbits with localized proximal tibial osteomyelitis (confirmed radiographically as Grades 2-4) were separated into six study groupss. Group 1 (control group): infected be ut left untreated for the duration of the study. Group 22: rabbits were treated for 4 weeks wi th subcutaneous vancomycin at 30 mg/kg twice= daily. Group 3: rabbits were treated for 4 weeks with subcutaneous vancomycin at 3 0 mg/kg twice daily plus oral rifampin at 40 mg/kg twice daily in 0.5% methylcellulose. Group 4: rabbits were treated for 4 ~weeks with subcutaneous tigecycline at 14- mg/kg twice daily. Group 5: rabbits were treated for 4 weeks with subcutaneous tigec=ycline at the same dose as in the rabbits in Group 4, plus oral rifampin at 40 mg/kg twic e daily in 0.5% methylcellulose. Rabbits receiving oral rifampin (Groups 3 and 5) wer—e given an oral nutritional supplemert (Ensure Plus®,

Abbott Laboratories, Columbums, Ohio) and a Lactobacillus spp. pre=paration (Kvvet

Supply, 3190 NRoad, David City, Nebraska) daily. Group 6: rabbi®s were treated for 1 week with subcutaneous tigecwycline at the same dose as in Group 4 ,, but were sacrificed 3 hours after administration of the last dose. At that time, blood and snfected bone samples were collected and tigecycline concentration was determined. Groups 1 to 5 were left untre=ated for 2 weeks after treatment phase of the experiment and sacrificec at 8 weeks after infection.

Rad® ographic Assessment

Radiographs of bilateral tibias were taken at initiation of therapy (2 weeks after infecztion), at the end of antibiotic therapy (6 weeks after infection), and at sacrifice (8 wee ks after infection). Radiographs were scosred according to a visual scales (Table 1) by three investigators, each blinded to the treatranent group, and the grades wemre averaged.

TABLE 1

Criteria for Radiographic Osteomyelitis Severity Grading in Rabbits

Description of Changes : 0 Normal, no chan ge compared with right tibia 1+ Elevation or disruption of periosteum, or both; soft tissue swelling < 10% disruptiom of normal bone architecture 10 - 40% disruption of normal bone architecture > 40% disruption of normal bone architecture *Visually estimated percenta ge of disrupted bone.

De=termination of Serum Levels of Antibiotic

Peak and trough levels of antibiotic were determined for Groups 2= and 4 at 1 hour (peak) and 12 hour (trough) after the initial antibiotic administration. See= Figures SA and 5Bs. Antibiotic concentrations were determined by means of a bioassay. _Antibiotic diffusion assay was performed as described above. Concentrations of antzibiotic were extrapolated from the respective standard c urves.

Determination of Bacterial Concentration joer Gram of Bone and Bone VE arrow

After sacrifice, gross cultures were performed for right and left titbias. Quantitative cosunts of S. aureus, in CFUs per gram, of Left tibial bone and marrow we=re determined for al3 study groups.

Culture Preparation: The bone marrow and the intramedullar—y canal of bilateral tibias were swabbed with sterile cotton tip applicators for gross cultures analysis of left tibias and quality assurance checks of right tibias. The inoculated apyolicator was streaked onto blood plates and then placed into 5 ml of sterile TSB. The platess and tubes were then 5S incubated at 37°C for 24 hours and growth and/or turbidity was recoreded.

The bone marrow was placed into a sterile, 50 ml centrifuge tmube and weighed.

The bone fragments were brokem into 0.5 cm’ chips, placed into a sterile, SO ml centrifuge tube, and the final product weighmed. Normal sterile saline, 0.9%, wass added ina 3 to 1 ratio (3 ml saline/gram of bone ovr marrow) and the suspensions were vortexed for 2 minutes. Six ten-fold dilutions of each suspension were prepared witch sterile, normal saline, 0.9%. Twenty-microliter samples of each dilution, including ~the initial suspension, was plated, in triplicate, onto blood agar plates and incubated at 37°CC for 24 hours. CFUs were counted at the greatest dilu tion for each tibia sample. The S. atmtreus concentration was calculated in CFUs per grarn of bone or bone marrow. The calcwmulated resultant was multiplied by 3 for bone samples and by 4 for bone marrow, in order— to account for their initial dilutions in saline and for the adsorption of marrow into the samline. The mean log of the S. aureus concentration for ezach was calculated. :

Statistical Analysis of Experimesntal Data

The standard deviation a.nd standard error of the mean were calculated for all raw data, including disc diffusion measurements, weight variances, radiomgraph grades, and bacterial counts. Linear regress ion analysis, least squares method, were performed for the antibiotic diffusion standard curves using the base ten log of the anti_biotic concentrations to plot the concentration (in pg/ml) versus the zone of inhibition me=asured (in millimeters). All subsequent di ffusion measurements were extrapolaated to micrograms/milliliter of antibio tic concentration from the standard curve utilizing the slope and y-intercept values der-ived from the least squares calculaticons.

Minimum Inhibitory Concentra tions and Minimum Bactericidal Cormcentrations

For the strain of methici llin-resistant S. aureus (inoculum of 10° CFU/ml) used in the study, the minimum inhibitory concentrations and minimum bac=tericidal concentrations for tigecycline were less than 0.2 pg/ml and 0.2 pg/ml, respectively. The minimum inhibitory concentration and minimum bactericidal concentration levels for vancomycin were 0.39 pg/ml and 0.78 pg/ml, respecte vely, yielding MIC/MBC ratio of 0.5. The minimum inhibitory concentration and minirmum bactericidal concentrations levels for rifampirm were 0.78 pg/ml and 1.56 pg/ml, respectively, yielding a ratio of 0.5.

DrugKinetic Leve=ls in Bone and Serum

All concern trations of antibiotic were derived f Tom the respective standard curves.

The logarithmic trends of the concentrations of tigecy~cline (14 mg/kg twice daily) in th ¢ sera of uninfected animals group are shown in Figure 1. The tigecycline, as depicted im

Figure 1, eliminated slowly, maintaining a steady level higher than MIC (0.2 pg/ml) by~ 12 hour (trough). Peszaks and troughs of tigecycline (14 mg/kg twice daily) and vancomycin (30 mg/kg twice dRaily) in the serum of infected rabbit_s after administration of the respective drugs a re shown in Figures 5a and 5b. The bone concentrations of tigecyclire (14 mg/kg, Bid) ir the infected rabbits group were measured separately in the infected tibia at the end of treatment, in which they averaged (0.78 pg/ml +/- 0.01 pg/ml, and in the uninfected tibia, im which they averaged 0.49 ug/ml +/- 0.01 pg/ml. The difference wa_s statistically signif=icant (p < 0.05).

Radiographic Finelings

A stage 2 to 4 osteomyelitis, according to Tabwle 1, was induced in all the infected animals. The init-al radiographic grades were similar between the groups. The averages grades for tigecyc=line, tigecycline + rifampin and varacomycin + rifampin groups at t=1 4 days were significantly greater than the average gradess at t=56 days (p<0.05). The cormtrol group showed the= least amount of improvement radiographically (0.2 +/- 0.2 or 9.1%), when compared vwith vancomycin (0.5 +/- 0.2 or 25%5), with tigecycline (0.9 +/- 0.1 or 40.9%), with vancomycin + rifampin (0.9 +/- 0.1 or 40.9%) or with tigecycline + rifammpin (0.8 +/- 0.1 or 400%) groups.

Figure 2 depicts the average radiographic sev=erity for each group at t=14 and t=36 days. Atthe end of the study (t=56 days), average ra diographic grades were compared. between differentz groups. The average grades for tigzecycline group, tigecycline + rifampin group ard vancomycin + rifampin group at ~t=56 days were significantly lowe=r than the average grades for the control group at t=56 days (p < 0.05). The key for figu-re 2 is as follows: Control = control group, no treatment; Vancomycin = subcutaneous vancomycin treat-ed group; Van + Rifam = subcutane=ous vancomycin with oral nfampixn treateed group; Gar-936 = subcutaneous GaT-936 treated group; Gar + Rifama = subcu. taneous Gar-936 with oral rifampin treated group.

Bone Cultures

A high percentage of tibias from urtreated infected controls (n=15) revealed positi ve cultures (80%) for methicillin-resi stant Staphylococcus aureus at a mean concentration of 9.21 x 10° CFU/g bone. "When compared to untreated controls, the vancomycin group (n=11), tigecycline group and tigecycline + rifampin group all demo=nstrated a significantly lower percentage of positive methicillin-resistzant Staplmylococcus aureus infection. In the vancomycin group, or 2 out of 11 =samples (18.2%) were positive for MRSA, and the average bacterial concentration Of the group was L 4 x 10> CFU/ gram bone (p < 0.05). In the tigecycline group, 1 out =f 10 samples was positive for methicillin-resistant StapFaylococcus aureus and the averagme bacterial concentration in the group was 20 CFU/ gwram bone, which is lower than eisther the controls or the vancomycin group (p < 0.05). One rabbit in vancomycin + rifampin group showed highe=r bacteria concentration than the control. The rabbits receiving tigecy~cline + rifampin treatment group demonstrated co dmplete eradication of bacteria from the tibia (0.0

CFU# gram bone in all the samples). Figure 3 compares the CFU/gram marrow and bone betwe=en all groups. Figure 3 demonstrates that tigecycline and tigecycline in combination with rifampin were an effective treatment for infection of the bone and infe=ction of the marrow with respect to controls.

The key for figure 3 is as follows: Control = control group, no treastment;

Vanc-omycin = subcutaneous vancomycin treated group; Gar-936 = subcutzaneous Gar-936 treate=d group; Vancomycin + Rifampin = subcutaneous vancomycin with oral rifampin treated group; Gar-936 + Rifampin = subcutaneous Gar-936 with oral rifanmpin treated group.

Advesrse Events

Of the 66 infected rabbits, a total owf 6 died before completion of tre atment. Of the 5 rabbits that died in the tigecycline treatment group, one of them was euthuanized at day 19 because of severe impairment of nutritmonal status. Another rabbit died at the day 17 of tigecycline treatment due to gastroenteroceolitis. Three of the rabbits in thiss group died at day 28 due to gastroenterocolitis and intol erance to anesthesia. One rabbis in the

! tigecycline + rifampin group died during treatment at day 15 due to~ gastroenterocolitis.

The gastroenterocolitis was rmost likely caused by alteration of the mormal flora of the large intestine. All rabbits w~ere monitored weekly for weight variawnce. The control group showed the greatest mean ga in (0.58 kg +/- 0.27), vancomycin the ssecond greatest (0.39 § kg +/- 0.26), vancomycin + rifampin group the third (0.21 kg +/- 0 32). Tigecycline group (-0.05 kg +/-0.32) and tigecycline + rifampin (-0.39 +/- 0.31)® group both lost weight after the antibiotic treatment_ Nearly all rabbits in the tigecycline gzroup and tigecycline + rifampin group presented with mild to severe symptoms of gastric dysfunction approximately 1.0-1.5 weeks post-antibiotic initiation, including de=creased appetite, dehydration, diarrhea, and/or weight loss. Figure 4A and B show tne weight variances between all the groups. The key for figures 4A and B is as follows = Control = control group, no treatment; Vancomycin = subcutaneous vancomycin treated group; Vanco +

Rifampin = subcutaneous vancomycin with oral rifampin treated gr-oup; Gar-936 = subcutaneous Gar-936 treated group; Gar-936 + Rifampin = subcut aneous Gar-936 with oral Rifampin treated group.

As for the safety, a higher number of deaths and side effectss were seen in the groups of rabbits treated with tigecycline. Enterocolitis due to tigecycline may be caused by an extensive destruction of the normal microbial flora of the bov=vel. The symptoms were attenuated by the administration of oral probiotics. The broad_ antimicrobial spectrum of tigecycline, in contrast with the narrower spectrum of \wancomycin, may help explain the difference observ-ed between the treatment groups.

Results

The count data for each animal in each tissue are listed in Table 2. The counts in the table are averages of tripl icate measurements made on each tissiae. Inspection of the data in Table 2 reveals that in treatment groups treated with test arti cles, the counts in most or all animals were 0. In the control group, non-zero counts were nrmeasured in marrow from 5 of 15 animals and in bone from 11 of 15 animals. There wass considerable variation in the magnitude of the non-zero counts in the control gromups, especially for bone.

The number of positive and negative cultures in each treatm: ent group, and the p- values resulting from comparisons to control were was follows:

TABLE 2

Count of Colony Formimg Units Per Gram of Bone and "arrow from Osteomyelitis

Study In Rabbits

Treatment Group | Counts (CFU/gm) | Counts (CFU/gm)

Eid il hl wenn | 0 |] 2 oo |v] + 1 1°

IE CR EE eo me mw mw

IE I EC

IE DC EE

EEC BC EC

I IT IC

IC ELI EN

Teer | 0 | mss

I EC ER

IEE EL ER

IE LE EC

IEE EL EC

6 [oo 1°

IEA EL CE

IL EC BC

IEE EC wo 0° I 0

Treatment Group | Counts (CFU/gm) | Counts (CFU/gm)

Marrow~ Bone

IEE EL

IE EL

IE EL

IEE EL

IEA I

IEC LE

EE

IE

IE

IEE

IE

IE

+ [oo 1 oo |]

EL CE

EL

Treatment Group | Counts (CFUJ/gm) { Counts (CFU/gm)

Marrow’ Bone

LC

530,000,000 1,040,000

IEE A A RA

IER RL

IE

IRAE EL

IE EC

Data from Rabbit osteomyelitis comparison o¥ tigecycline, vancomycin, and rifampin

In marrow, the proportion of positive cultures in the tigecycline and tigecyscline + rifampin treatment groups was 0, which in cornparison to the proportion of 0.33 (5/15) in the cortrol group was almost statistically significant (p=0.06) at the conventional go=0.05 level. The proportions in the vancomycin and vancomycin + rifampin groups wer-€ not statisti cally significantly different from the coentrol group. In bone, the proportion of positive cultures in each of the groups treated with test articles was statistically signifi cantly lower than the proportion in the contro! group.

In an animal model of methicillin-resi stant Staphylococcus aureus, endocarditis, 14 mg\kgs bid tigecycline was shown to be more effective than 40 mg\kg vancomycir= (Murpehy, Antimicrob Agents Chemother 2000; 44(11): 3022-3027). In a rat model, dosagees as high as 80 mg\kg\day were admin istered. However, in the rabbit modeel used hereima, the administration of dosages higher than 14 mg/kg per day caused relevamt morbidity and mortality in the animals (data mot shown). Therefore, the above citaed dosag e was used in this study. Even though the goal was not to study the pharmacokinetics of tigecycline in rabbits, some drug levels measurements were performed in order to ensure that an adequate dosage was being used in the animal model.

The d_ata confirm that drug levels in serum were still above the MIC of the staphylococcal strain used 12 houurs after the last administration. Moreover, the drug has displa-yed a relevant bone permetration, and therapeutic levels of ti gecycline have been found in the infected and unin fected bone. The higher concentration of drug found in the in=fected bone is another relevart finding, which requires further stumdy.

EXAMPLE 2: Distribution of Tigecycline in Human Tissue after One Intr—avenous

Administration of 100 mg.

This exarmple shows the penetration of selecteed tissues in human subjec ts after a single intravenoums administration of tigecycline. The= data demonstrate a rapid distribution phase, with a proslonged half-life and a high volume eof distribution at steady state. They further establish the penetration of bone, synovial fliaid, lung, gall bladder, and colon in human subjects. Penetration improves treatment of toone and joint infections.

Studies o=f the pharmacokinetics of intravenous tigecycline in humans hmave shown that there is a rapid distribution phase, with a prolon ged half-life (40 to 60 hou=xs) and a high volume of Clistribution at steady state. Animal =studies with radiolabeled tmgecycline suggest that this rapid distribution phase and high volume of distribution at ste-ady state represent penetration of tigecycline into tissues incluading lung and bone. Sprague-Dawley rats (18 males) vere given carbon-14 tigecycline at =a dosage of 3 mg/kg by 30 -minute infusion. Conce=ntrations or radioactivity were determined in tissues of 3 rats/time point at the end of the in fusion and at 1, 8, 24, 72, and 168 h_ours after the end of infusi_on. For all tissues, peak radkioactivity concentration were obser=ved at the end of infusion. In general, radioactivity wa s well distributed to most tissues, with the highest concentraticons as follows: bone>Mone marrow>salivary gland, thyroied, spleen, and kidney. In e=ach of these tissues, the ratio= of area under the concentration-timme curve in tissue to area urader the concentration-timme curve in plasma was >10.

The objective of this study was to determine= the tissue and correspond ®mng serum concentration off tigecycline at selected time points mn lung, colon, gallbladder tissues, bone, and synowial fluid. Samples were taken from subjects scheduled for lune g, colon, gallbladder, or tone surgery, or a lumbar puncture who were given a single dose of tigecycline adm _inistered intravenously.

Pre-spec=ified tissue/fluid sampling of either lung, colon, gallbladder, beone, and synovial fluid was performed on each subject during surgery at 4 hours, 8 houmrs, 12 hours, or 24 hours afte=r the start of a single dose of 100 mg tigecycline administered over 30 minutes. Serum was collected from all subjects at hour 0 (before the first dose), approximately 30 minutes (end of infusion), and at the time corresponding to tissue/fluid collections. Tissue and seru m concentration was determined accoarding to the method set forth below.

Investigational Parameters for Serum Samples

Samples of human serum and tissue from study subjects who had received tigecycline were analyzed according to methods that had been previously validated.

Serum samples and synovial fluid (0.2 ml} were mixed with 0.6 nil internal standard in acetonitrile, the supernatant evaporated to dryness and the residue reconstituted in 200 microliters mobile phase. A.liquots (10 microliters) of the reconstituted samples were injected into an LC/MS/MS .

The data was acquirezd by and analyzed on PE SCIEX “Aralyst” version 1.3 software. Linear regression , with 1/x? weighting was used to obtzain the best fit of the data for the calibration curves. T he lower limit of quantitation was 10 ng/ml for serum and synovial fluid samples, 10 mg/g for the colon and gall bladder sanrples, and 30 ng/g for the bone samples.

Quality control samples (2 sets) at low (25 ng/ml), mediurm (500 ng/ml) and high (1500 ng/ml), prepared in hwiman serum, were analyzed with eacla set of serum samples.

For colon, gall bladder and Rung samples, two sets of quality cont rol samples at 25, 500, and 1500 ng/g were analyzed with each set of tissue samples. For bone, two sets of quality control samples at 1900, 500, and 1500 ng/g were analyzed with each set of tissue samples.

The curves were linear in the range from the 10 to 2000 ng/ml for serum and synovial fluid and from the lower limit of quantitation to 2000 ng_/g for tissues. A run was considered successful if no amore than two quality control sampless were outside the range of 85-115% of target and no two quality control samples at the sa_me concentration were outside that range. If two quality control samples at the same coracentration were outside that range, only concentrations between the remaining quality coritrol samples were reported.

Materials an«d Methods for Serum Samples

Tigeccycline was measured in human serum using an LC/MS/MS method. The primary stoc-k solution of tigecycline was prepared at 1 mg/ml by dissolving in methanol.

A secondary stock solution was prepared from the primary stock solution by dilutmngto a concentration of approximately 40,000 ng/ml with acetonitrile. The stock solutiors were stored at —20 °C when not in use. A primary internal standard solution of tert-buty1-d9- tigecycline vovas prepared at a concentration of 1 mg/ml in methanol. A secondary internal standard stoeck solution was prepared by diluting th. e primary stock solution to a concentratio n of 100 micrograms/ml in acetonitrile= with 0.1% trifluoroacetate add ed. The primary and secondary stock solutions were stored at 20 °C. The working internal standard wass prepared by diluting to volume with zZacetonitrile/0.1% trifluoroacetatze. The working intezmal standard was stored at 4 °C when not in use. On the day of analy=sis, the secondary stock solution was brought to room temperature before use to prepared the standard curve working solutions. The standard cumrve was prepared at approxima tely 2000, 1600, 1000, 500, 250, 100, 50, 20, and 10 ng=/ml by serial dilution in blank Fhuman serum.

The eextraction procedure was as follows: t«o 200 microliters of calibrator, quality control or smmple was added 600 microliters of intemal standard working solutiorm and vortex mixeud. The samples were centrifuged for 140 minutes at 13000 rpm to sepa rate the layers and the supernatant was transferred to a culture tube. The samples were evaporated to dryness ira a Speed Vac. The residues were reco nstituted by sonicating in 200 microliters Of mobile phase and 10 microliters was injected in the LC/MS/MS.

The LC/MS/MS was composed of HPLC (Agilent 1100), Mass Spectrome=ter (Applied Biosystems AP13000), Column (Aquasil «C18, 50 x 2.1 mm i.s., Smicron (ThermoKewystone) with mobile phase of 16% aceteonitrile, 6% methanol, 78% wamer, and 0.1% tetreflwioroacetate, flow rate approximately 0_35 ml/min, injection volume 10 microliters, Detector Conditions: 119 scans in period, MRM scan type, positive p-olarity, turbo ion spray source, at low resolution, using nitrogen at 6 psi as a nebulizer gass, a curtain gas, and a collision gas, with ion energy at <4500 mv, and ionspray temperamture at 450°C. The= detector monitored tigecycline and thes internal standard.

Samypples were analyzed over three analytical runs. On each day of sample analysis, a complete sstandard curve was run, along with quality control samples and study subject samples. Samples that had a measured concentratieon greater than the highest calitorator were diluted by mix ing 100 microliters sample with 900 microliters blank humar— serum and analyzing 200 mnicroliters of the mixture as previo usly described. Quantificamtion of tigecycline in serurrm was achieved by comparison to a standard curve prepared ir the appropriate matrix 2nd calculated using a (1/concentramtion)® weighting factor.

The limit of” quantitation for tigecycline was 10 ng/ml. No peaks interfering with the determination o f any of the tigecycline isomers were detected in any of the p-re-dose samples. All calibrators and quality control samples vere within range (85-1154 of target). Results of ssamples are presented in Table 1. “Results of standard curves and calibrators are preseented in Table 4.

Investigational Parameter for Tissue Samples

Stock solut®mon and internal standard solution was prepared as per investi gation parameters for sertmm samples above. The standard curve working solutions were prepared at approximately 1%0000, 8000, S000, 2500, 500, 250, 100, and 50 mg/ml. On the day of analysis, 40 microl iters of the working solutions were= added to 200 mg of tissue to produce calibrators at 2000, 1600, 1000, 500, 250, 109, 50, 20, and 10 ng/ml. Canine tissue was substitu-ted for human tissue to prepare thes calibrators and quality comtrol samples. Because of the limited availability of canin_e gall bladder, canine colon was used to prepare the stanedard curve for the analysis of humzan gall bladder. Colon wass shown to be an appropriate ssubstitute matrix for the analysis of gall bladder samples.

The extrac®ion procedure was as follows: to 2200 mg of calibrator, quali ty control or sample was add_ed 3 ml of internal standard working solution and samples w ere homogenized usin ga hand homogenizer. The samples were centrifuged for 10 minutes at 14000 rpm to sepamrate the layers and the supernatant was transferred to a centri_fuge tube.

The samples were evaporated to dryness in a Speed “Vac. The residues were reaconstituted by sonicating in 2€00 microliters of mobile phase and 10 microliters was injecte=d into the

LC/MS/MS. LC/TMS/MS conditions were the same aas those used to analyze se-Tum samples. Synovia_l fluid samples were extracted in thhe same manner as serum samples.

Samples were analyzed over several analytic al runs. On each day of sa_mple analysis, a complete standard curve was run, along vevith quality control sample=s and tissues. The stancdard curve was prepared in the subsstitute matrix appropriate teo the tissue samples being analyzed. Samples which had a measured concentration greater— than the highest calibrator (200 ng/g) were homogenized wit“h internal standard at 10 or— 20 times the concentration use for the stanclard curve. An aliquot (300 microl iters) (10 fold dilution) was evaporated to dryne=ss and the samples were reconstituted so that the peak area ratios and peak areas were within the range of the standard curv-e.

Quantification of tigecycl-ine in tissues was achieved by com=parison to a standard curve prepared in the appropriate matrix and calculated using a (1/concentration)’ weighting factor. For synovial fl-uid, the calibrators were prepared imn phosphate-buffered saline. A second set of calibrators was prepared in an artificial synoevial fluid composed of the following components: 100 rnmol/L glucose, 2.03-2.26 g/L hyaluronate and approximately 8 g/L albumin adjusted to pH 7.4. The calibration cumrve prepared in PBS and the recovery, a correction factor was calculated by performing am linear regression of determined concentrations of arta ficial synovial fluid samples from he PBS curve verses the theoretical concentration of those samples using a power equation (y=y0 +ax®).

Because the determined concentration of study subject samples was- in the low range of the calibration curve, only the calibr ators from 20 to 500 ng/ml were us=ed to calculate this regression. The results of this rezgression showed a strong correlaticon (r* = 0.9996) and back-calculated concentrations ofthe ASF calibrators were betweern 94 and 122% of their target values over the complete range of the standard curve (20-200=0ng/ml). The regression equation was then applied to the concentrations of study subject samples from the PBS standard curve and the corrected concentration of tigecycli—ne in synovial fluid samples was determined.

The limit of quantitation for tigecycline was 10 ng/ml. Meamsurable concentrations of tigecycline were found in all mmatrices analyzed. All calibrators &and quality control samples at concentrations similar to the samples were within range (85-115% of target).

Results of samples are presente in Table 4 (tissues) and Table 5 (s- ynovial fluid).

Results

The data demonstrate a rapid distribution phase, with a prol.onged half-life and a high volume of distribution at steady state. They further establish the penetration of bone, synovial fluid, lung, gall bladdeT, and colon in human subjects. Additionally, concentrations in synovial fluid show rapid distribution and prolon ged retention of tigecycline as compared to data from serum at similar times.

TABLE 3

Results of Serum Analysis

Calculated Calculate ad Calculated

Sample Concentration Time Sample Concentration Time Samm pie Concentration Time 1D. {n&mill Hour 1.0. (np)miZ. Hour I) fap Imi Hour

IA BQL 0 2A BQL 0 40A BQL_ 0

IB 5450 0.5 28 1810 0.5 40B 195@0 0.5

Ic sis 24 1c 251 4 40C 80.65 24 2A BQL [V] 23A BQL 0 41A BQEL 4} 2B 1080 0.5 38 1530 0.5 4B 258 © 0.5 2 15 4 23¢ 74.4 24 aC 191 12 aA BQL 0 2A BQL 0 42A BQ'L 0 4B 1490 0.5 24B 1650 0.5 428 789= 0.5 ac 175 4 uC 85.6 12 42C 1982 8

SA BQL 0 25A BQL 0 43A BQ L I

SB 1100 0.5 5B 3550 0.5 43 11580 0.5

C16 12 25C 136 4 a3C 77. 4 24 6A BQL ° 26A BQL 0 a4A BQwL 0 68 1170 0.5 268 878 0.5 44B 9s 0.5 6C 113 12 26C 120 12 44C 143 4

JA BQL 0 271A BQL 0 45A BIL 0

IB 1640 0s 27B 847 0.5 458 85a 0.5 ic 917.2 12 27C 78.9 12 45C 299 4 8A BQL ° 2A BOL 0 46A BOL ) 8B 1710 0.5 238 922 0.5 46B 14°20 0.5 8C 186 4 28C 120 12 46C 66 .6 24 9A BQL 0 294 BQL 0 47a BOL °

SB 1860 0.5 29B 5190 0.5 47B 44 7 0s sc 221 a 29C 147 4 47C 25 2 4 10A BQL ° 304 BOL 0 48A BCL °o 10B 27400 0.5 308 1190 0.5 488 97 6 0s 10C 244 a 30C 50.8 24 asc 86m.9 24

HA BQL 0 3A BQL 0 49A BCL 0 1B 1320 0.5 3B 2320 0. 498 1200 0.5 1c 47.2 12 iC 166 4 a9C 102 12 132A BQL ° 32A BQL 0 S0A sel 0 12B 6950 0.5 3128 3550 0.5 SOB 12830 0.5 12C S44 24 32¢ 42.1 24 50C 1536 8 15SA BQL 0 3A BQL ) S1A BeQL 0 40 15B 1960 0.5 338 620 0.5 51B 1=26 0.5 15C 250 4 13C 43.7 24 51C 427 24 16A BQL 0 334A BQL 0 52A B QL 0 16B 741 0.5 348 4080 0.5 52B 82 0.5 16C 107 12 34C 92.7 12 s2¢ 1228 a 45 174 BQL 0 35A BQL 0 S3A B-QL 0 178 1110 0.5 3$B 2430 0.5 s3B 1 060 0.5 17¢ 51 24 35C $3.6 24 53C 2.09 4 183A BQL 0 36A BQL ) S4A B=QL 0 188 761 0.5 368 2300 05 54B 1 850 6.5 50 15C 133 8 36C 65.7 24 s4C 2 06 8 194 BQL 0 37A BQL ) SSA PRQL a 198 1240 0.5 378 2415 0.5 SSB 6% 28 a.s 19C 162 4 37C 95.7 12 55C q-31 4 20a BQL 0 38A BQL 0 55 20B 903 0.5 188 4600 0.5

20C 106 12 38C 106 12 21A BQL 0 A9A BQL 0 21B 870 0.5 98 5130 0.5 21C 778 4 39C 342 4 -— ' BQL = below quantitative limits

TABLE 4

Results of Tissue Analysis Calculated *BQL = below quantitative limi ts of the assay (<33.2 ng/ml)

Sample Concenmtration

LD. (n2/2) Tissue 001 8210 Gall Bladder 002 1560 Gall Bladder 004 41.6 Bone 005 20700 Gall Bladder 006 46.5 Bone 007 33.3 Bone 008 79.3 Bone 009 1890 Lung 010 141 Bone 011 7640 Gall Bladder 012. BQL Bone 015 8400 Gall Bladder 016 824 Gall Bladder a7 933 Bone 018 3750 Gall Bladder 019 18900 Gall Bladder 020 269 Bone 021 86.6 Colon 022 50.0 Bone 023 1180 Gall Bladder 024 BQL Bone 025 1550 Gall Bladder 026 91.2 Colon 027 BQL Bone 028 598 Colon 029 3240 Gall Bladder 030 BQL Bone 031 5960 Gall Bladder 032 938 Gall Bladder 033 BQL Bone 034 3480 Gall Bladder 03S 778 Gall Bladder 037 3850 Gall Bladder 038 BQL Bone 039 198 Colon 40 040 1500 Gall Bladder 04) 106 Colon 042 238 Gall Bladder 043 995 Colon 044 725 Colon 45 045 814 Colon 046 BQL Bone 047 453 Colon 048 BQL Bone 050 618 Colon 50 051 653 Lung 052 35.5 Bone 053 BQL Bone 054 36.1 Bone 055 18 Colon 55 _—

TABLE 5

Results of Synovial Fluid Analysis

Calculated

Sample Concente—stion Time 1.D- (ng/ml) (Hour) 4 39.-9 4 6 62. 8 12 8 152 4 130 4 10 12 46. 4 24 20 1 12 22 187 4 24 65-0 12 30 37-8 24 3 25..9 24 38 65. 7 12 46 55 .4 24 4B 45 0 24 52 70 .6 4 se 70-.9 8

EXAMP®LE 3: Tissue Distribution in RRats Treated with Tigecycline

This study was conducted to quan +titate ['*C]-tigecycline-derived raclioactivity in tissues by whole body autoradiography ussing phosphor imaging, following a single 30- minute 3 mg/kg intravenous infusion of [ **C]-tigecycline to male Sprague-Eawley and

Long-Evans rats.

Material=s and Methods

T igecycline was supplied by the Analytical Department, Wyeth-Ay erst Research,

Montrea 1, Canada. ['*C]-tigecycline was supplied by Amersham (Boston, IMA).

Radiocheemical purity and specific activit-y of bulk ['*C)-tigecycline was 9&% and 93.6 microCi~mg, respectively.

SSterile water was used to make th e intravenous dosing solution. Thae liquid scintillation cocktail used in counting the= radioactivity in plasma and urine was Ultima

Gold (Paackard Instruments Co., Meriden, CT). £2 Model 3078 Tri-Carb Sample Okxidizer equipped with an Oximatze-80 Robotic

Automa=tic Sampler (Canberra-Packard CCo., Downers Grove, IL) was used for combustion of blood samples. Permafluor E liquid scintillation cocktail (Packard Instruments Co.,

Meridarm, CT), Carbo-Sorb-E (Packard Irmmstruments Co., Meridan CT) carbeon dioxide 40 absorbemr and de-ionized water were used to trap radioactive carbon dioxide generated by

! combustiz on of the sample in the oxidizer. Blood aliquots were transferred to combusto- cones aned cover pads (Canberra-Packard Co., Downers Grove, IL) for comb-~ustion.

Mall radioactivity determinations (dose , blood and plasma) were made= using a To-

Carb Mosdel 2700 TR liquid scintillation counter (Canberra-Packard Co., Downers Grove,

IL) with an Ultima Gold or toluene standard curve. Counts per minute (CPIM) were converte=d to disintegrations per minute (DPM) by use of external standards of known radioact®vity. The quench of each standard was determined by the transforrmed spectral index of "an external radioactive standard (TS IE). The lower limits of detec@ion were defined as twice background.

Male Sprague-Dawley and Long-Evans rats were obtained from Charles River

Breedin_g Laboratories, Raleigh, NC, and were quarantined for at least one waweek prior to the start- of the study. The intravenous dosing solution (1.02 mg/ml) was prepared by dissolving 6.90 mg of unlabeled tigecycline and 5.30 mg of [“C]-tigecyclire in 12 ml sterile water. The dosing solution was diluted and radioassayed directly in Ultima Gold scintillaation counting cocktail (Packard Inc.) . All determinations of total ramdioactivity were m=ade with a Packard 2700 TR liquid scintillation spectrometer (Canb erra-Packard

Co).

The rat body weights ranged from 0.206 to 0.301 kg. All rats received a single 30 minute intravenous infusion dose of ['*C]-tigecycline via a jugular vein carula, (3 mikg, 3 mg/kg =s active moiety, 40 microCi/kg) usirag a Harvard infusion pump 22 (Harvard

Appara_tus, Southnatick, MA). All pumps were calibrated prior to the administration of the compound. Rats were anesthetized with. isoflurane prior to cardiac purmcture gxsangmuination at the prescribed times after dosing. Sprague-Dawley and “Long-Evans rats were saxcrificed one per time point at 0.5, 8.5, 24, 72, 168 and 336 hr post-close.

Control whole blood was collected from male Sprague-Dawley ratss into tubes contairming sodium heparin. Pooled blood wras used to prepare the calibratmon standards and quaality control samples. The standards were used to construct the staradard curve for the quantification of radiolabeled drug distribution in tissues of whole blood cryosections.

The quality controls, which were embedded in the same CMC block with seach rat, were used foor assessing intra- and inter-section variation in the thickness of rat whole-body cryosesctions.

A 200 microCi/ml stock solution of ['*C]-glucose (New England Mluclear, Boston,

MA) was serially diluted with whole blood from male Sprague-Dawley ra_ts to obtain fourteen stan dards at the following concentrations: 832, 485, 250, 122, 48.6,24.3,1220, 4.72, 2.36, 0_853, 0.638, 0.405, 0.327, and 0.221 nCi equiv./ml. The low, mid and high

GCs concentrations were 12.39, 25.9 and 508 nCi eequiv./ml.

Immediately following euthanasia, each rat was totally immersed in a bath off hexane and cry ice (-75 °C) until frozen. Each carcass was dried and stored at ~30 °CC until embedded. Each animal was embedded in a mold €15 cm x 45 cm) by adding low viscosity, 18% carbosymethylcellulose (CMC) and frozen by placing the stage in a hexane-dry mce mixture.

Froz-en blocks were transferred to the Jung <Cryomacrocut 3000 (Leica Instruments

GmbH, Nus sloch, Germany) and allowed to equilibrate overnight to the cryotome irternal temperature for at least 12 hours before sectioning...

Each frozen rat was sagitally sectioned at —20 °C. A sufficient number of sections were collected to ensure sampling of all tissues of interest. The sections were dehycirated overnight ira a cryochamber and then rapidly transferred to a dessicator containing (CCaSO4 to prevent c-ondensation of atmospheric moisture vwhile equilibrating to room tempe rature.

The section s were mounted on cardboard and labe led with ['“C]-labeled black ink vith a unique identification number. Radioactive ink wa_s prepared with equal volumes of India

Ink and ['*C]-CL-284846 (100 microCi/ml). A small piece of Scotch tape was placed over the dried ra_dioactive ink to prevent the contamination of the storage phosphor screens.

Pho- sphor imaging plates, BAS-SR 2025 (E'uji Photo Film Co., Japan) were exposed to bright visible light for 20 minutes usin g an IP eraser (Raytest, USA Inc. , New

Castle, DED) to remove background radiation. Sections and calibration blood standards were concumrrently placed in direct contact with Ip s and exposed for 7 days. All sections were storec] at room temperature in a lead shieldirag box to minimize background le=vels.

Phosphor images were generated using a Fujifilm BAS-5000 Bio-Imaging Analyzesr and quantitated by MCID M2 Software, version 3.2 (Imaging Research Inc., St. Catherines,

Ontario, Caanada). The STDs and QCs were analsyzed using the circular sampling t ool in the MCID software program. Areas of interest in. whole-body sections were manuzally outlined w ith the regional sampling tool to generate count data.

Racdioactive concentrations in select tissues were determined by digital analysis of the resultirg autoradiograms on the basis of a caldbration curve. A calibration curve of stated concentrations (nCi/g) verses MCID respomse, photostimulated li ght/mm? (E2SL/ mm?-mimums background converted to nCi/g) for esach standard was generated by weighted

(1/x%) linear rregression analysis. The linear regressi on curve was then used to determine the concentration of unknown radioactivity of study” samples. The regions of i-mterest (ROI) which visually exhibited levels of radioactivity were individually outlineed or autoscanned with sampling tools to obtain radioacti-vity concentrations. To de~termine the limit of quammititation for QWBAR, coefficients of v ariation from blood standards tested were determined, and the limit of quantitation was clefined as the lowest conce=ntration at which the comefficients of variation did not exceed 1 5%.

Plasrma aliquots were combined with 10 ml -of Ultima Gold™ scintillation counting cocktail (Packard Inc.) and directly counted. Blood samples were combusted using a

Model 307 ssample oxidizer (Packard Instrument Company). The resulting ['* ClO, was trapped in Czarbo-Sorb®, scintillation cocktail (PerrmaFlour®E+) was added, &and the samples wer—e quantitated by LSC.

Samples were counted in a Packard 2700 TR liquid scintillation spectr—ophotometer (Canberra-P®ackard Co.) for 10 minutes or 0.2 sigma. Counts per minute were converted to disintegrations per minute by use of a quench curvee generated from external standards of known radioactivity. The quench of each standard and sample were determimaed by full spectral shift. Limit of quantitation (LOQ) for LSCC was defined as two times= background.

The pharmacokinetic parameters for ['“C]-€GAR-936-derived radioact—ivity were calculated wmsing the intravenous infusion (Model 2202), Non-Compartment Aralysis

Module of ™WinNonlin, ver. 1.1, (Scientific Consul tants, Inc. Research Triang=le Park, NC), which appli es a model-independent approach and standard procedures as described in

Gibaldi andl Perrier. Gibaldi, Pharmacokinetics, 1982. In determining the mean concentratison, zero was substituted for any values that were below the limit ©f quantitation (5.10 ng eq uiv./g). For IV infusion dosing, C30 main was the concentration a~t 30 minutes, the first sarmpling time point. The maximum plasrma concentration (Cpa) aned the correspond ing time of peak concentration followirg I'V administration were aobtained directly by numerical inspection from the individimal concentration-time data. The terminal half-life was calculated by the ratio of In22/Az where Az is derived frcom the terminal sleope of the concentration time curve. TEne area under the plasma concentration versus times curve from zero to infinity was calcul ated using the trapezoidal rule, where

Clas is the TXast measurable plasma concentration. Tissue to plasma concentr=ation ratios were calcu lated according to the following equati@n: Cyssue/Cplasma, Where Cy; 55 equals the drug concentration in tissLae (ng equiv./g), and Cpusma €quals thes drug concentration in plasma (ng equiv./g).

The specific activi ty of [**C}-tigecycline (base) was deteermined by gravimetric assay to be 43.94 uCi/mg (Table 1). The concentration of the losing solution was 1.02 mg/ml. Animals received an average dose of 3.09 + 0.11/kg cosmpared to a target dose of 3 mg/kg.

Individual concentrations (ng equiv./g) of total radioact ivity in tissues of Sprague-

Dawley rats following a 3 mg/kg IV infusion of [*C]-tigecyclimne are represented in Table 6. Pharmacokinetic parameters in tissues are presented in Tabl e 7. Tissue to plasma ratios are presented in Table 8.

Individual peak coencentrations (Cmax) Of total radioactivity occurred at the end of infusion for virtually all o fthe tissues. Tissues with the highest concentrations of radioactivity were kidney (7601 ng equiv./g), liver (7300 ng eq uiv./g) and spleen (6627 ng equiv./g) (Table 7). The tissues with the lowest peak concentration of radioactivity were the brain (54 ng equiv./g) and eyes (108 ng equiv./g) (Table 7). Cay was greater than 2000 ng equiv./g for most (70%) tissues. Tigecycline-derived mradioactivity at Crux was lower in plasma than in al 1 tissues, except brain, eyes, fat and testes (Table 7). By 24 hrs, all tissues had higher concentrations of ['*C]-tigecycline-derivezd radioactivity (Table 6) than plasma except eyes.

Individual tissues «concentrations of radioactivity at 168 hours for most tissues declined to 1% or less, rel ative to their Cay, With the exceptior of bone, kidney, liver, skin, spleen and thyroid (Table 6). By 336 hours, most tissues had concentrations below the quantitation limit (5.140 ng. equiv./g) except bone, kidney, skin and thyroid. However, the concentrations in theses tissues (bone, thyroid, kidney and skin) were greatly reduced from Cmax. In general, tisssue concentrations of ['*C]-tigecyclin e-derived radioactivity in bone, kidney, skin and thyroid at 336 hours were 19%, 0.18%, 0/43% and 6% of Cys, respectively.

Using AUC as a measure of tissue burden, the bone andl thyroid had a much greater burden than any other tissues. The highest AUC values were ir the bone (794704 ng eq-hr/g), thyroid (330047 ng eq-hr/g), salivary gland (110979 ng eq hr/g), kidney (70704 ng eq-hr/g), thyroid (3304 7 ng eq-hr/g), spleen (70522 ng eq-hr~g) and liver (53527 ng eq-hr/g). The tissues with lowest burden were the brain (2865 ng eq-hr/g), fat (3500 ng eq-hr/g) and testes (10303 ng eq b/g). AUC exposure in bone was two-ttimes higher than the next highest tissue (thyroid). Tissue:plasma AUC ratio values were greater than one for the majority of the tissues (Table 7).

The terminal half-life for ['*C)-tigecycline-derived radioactivity scanged from a low of 5 hours in the fat to more than 200 hours in the bone and thyroid, cormpared with a plasma t,; of 24 hours (Table 7). Tissues with the longest elimination half-life were thyroid (804 hours), bone (217 hours), skin (182 hours) and kidney (118 hours) (Table 7).

The tissue:plasma concentration ratios (Table 8) were greater than one for the majority of tissues, with the exception of brain, eyes, testes, and fat at thes 0.5 and 8.5 hour time points. At 24 hours, all ratios were greater than one. The highest ti ssue to plasma ratios occurred for some tissues at 72 hours:bone (414), thyroid (56), skimn (19.3), spleen (16.7), and kidney (11.1). The bRood:plasma ratios were greater than one for all time points, suggesting that there was substantial partitioning of ["C]-tigecyc line-derived radioactivity into blood cells.

The distribution of ['“C]-ttigecycline-derived radioactivity to mel=anin-containing tissues (skin and uveal tract) in Long-Evans rats was also evaluated up tc 336 hours post- dose. Blood and plasma concentrations of [**C]-tigecycline-derived radi oactivity in Long-

Evans rats were similar to Sprague-Dawley rats (Tables 2 and 5). Peak radioactivity concentrations (Cmax) Were observed at the end of infusion (0.5 hour) for skin, uveal tract, plasma and blood (Table 9). The: Crux of ['*Cl-tigecycline-derived radio=activity in skin and uveal tract was 1997 and 2502 ng equiv./g, respectively. The AUC of ['*C] ['*C]- tigecycline-derived radioactivity in skin and uveal tract were 109296 andl 233288 ng equiv-hr/g, respectively. The terrninal half-lives for skin and uveal tract were 473 and 20 hours, respectively (Table 10). The half-life values are of questionable meaning since the elimination phases in the concentration-time profile could not be identified with certainty.

This is also reflected in the extrapolation of AUC data for uveal tract andl skin.

The tissue:plasma concen tration ratios were greater than one for skin and uveal tract at all time points (Table 7). The overall highest tissue to plasma ratios occurred at 72 hours in skin (179) and uveal tract (393). The tissue:plasma AUC ratios —were 8.45 and 18.0 for skin and uveal tract, respectively, and indicate that these tissues =selectively retain significant concentrations of [**C J-tigecycline-derived radioactivity. The= data suggest that radioactivity selectively partitioned in the melanin-containing region of thhe rat eye. Mean tissue concentrations of radioactivity at 336 hours for skin and uveal tract ceclined to 8 and 1%% of Cray, respectively.

Table 6

Mean Concentrations of Total Radioactivity in Tissues Following a Single 30 Minute

Infusion of ['*C)-Tigecycline in Male Sprague-Dawley Rats

EA LC CI LC KEIN Bc Rl

Adrenal 3580 941 68.9 19.3 <5.10 <5.10

Ee A

Bone 4376 1562 291 22.5 <5.10 <5.10 [OT

ELC EA RC Co Le ETL

REC Rc LLC EC 2 LEN

Lymph 3473 1276 29.1 <5.10 <5.10

FE a A

Salivary 5711 6313 300 31.6 <5.10 <5.10

Fl

I ELC LC I

Mable 7

Phar macokinetic Parameters of Total Rzadioactivity in Tissues Follovving a Single 30

Minuate Infusion of [**C}-Tigecycline in “Male Sprague-Dawley Rats

Tissume Type | Cmax Tin AUC AUC Tissue:Plasma

Nl eo A ey [A

Adre=nal 3580 13 29153 29515 2.77

Fl

Bones 4376 11 47116 47468 4.46 pre A A a

LE LCA EN cc Cc AN

Ro EA LCN cc LC CC

Lymph 3473 13 36478 37010 3.47 (ol I A [(Musde [2260 [8 [M83 [3000 [38

Paces [#437 Jo [308 [Bod [30

Pinay [363 [10 Mss [esies [424

Sal-ivary 6313 110558 110979 10.4 rr I

EC CEC LC Ec EJ EEN

Table 8

Tissue:Plasma R2atio Following a Single 30 Minute Infusion of (**C}-Tigecy<line in

Male Sprague-D=awley Rats

EA Lc LC EE CN LOS En

Adrenal 4.-00 1.87 4.67 4.47 NA ™NA

Ere A I

Bone 4. 89 3.10 19.7 5.21 NA NA oe [7

Lymph 3.88 2.53 12.2 6.75 NA NA oo

Ei EEC LE CC EC LS ECS

Salivary 6 .45 12.53 20.3 7.32 NA “NA

Fe ll A

Table 9

Mean Concentration (ng equmiv./g) of Total Radioactivity in Tissues Following a

Single 30 Minute Infusion of ~ ['*C)-Tigecycline in Male Long-E vans Rats

BAC EAC Le CE GL Fc

Table 10

Pharmacokinetic Parametemrs of Total Radioactivity in Tissuess Following a Single 30

Minute Infusion of [“C]-Tiggecycline in Male Long-Evans Rats

Tissue Type | Cmax Tin AUC AUC Tissue:Plasma re 4 fn ns

CL Ec LN Lc LA LJ ek EEC CU LE EI LL

Table 11 Tissue:Plasma Ratio Follovving a Single 30 Minute Infusion of {'‘C]-Tigecycline in

Male Long-Evans Rats

EA REC LEE LS KE LL

Discussion

The distribu_tion of radioactivity to tissues was evaluated following a single thirty minute intravenous infusion (3 mg/kg) of ["*C]-tigecyacline to Sprague-Dawley and Long-

Evans rats. Radioasctivity was distributed to tissues rappidly, with Cmax, observed amt the end of infusion (0.5= hr) for most tissues. Tissue concentrations were similar to a study conducted previous-ly by the tissue dissection method. The extensive distribution omf tigecycline into a variety of tissues is suggestive of a very large volume of distribution.

This finding confirms the previous observation of a hi gh volume of distribution in wrats and dogs. In general, tine elimination of radioactivity fromm most of the tissues was slower than the rate from plasm_a.

The concentrations of [*C]-tigecycline-derivead radioactivity in tissues of S prague-

Dawley rats was hi zgher than plasma at most of the time points. Tissue concentrations of radioactivity at 168 hours for most tissues decline to 1 % or less, relative to their en d of infusion values. Bxy 336 hours, concentrations in bone, thyroid, kidney and skin de=clined to 19%, 6.25%, 0.1 8% and 0.43% of Cmax values, respectively.

Tissues witkn the highest levels of exposure in Sprague-Dawley rats, as indiccated by the mean AUC valiaes, were bone, thyroid, salivary gl ands, kidney and spleen. The elimination half-liv-es were quite long (5 to 217 hours, with bone, skin and thyroid_ having the longest elimination half-lives. The value of half-1# fe for the thyroid tissue is questionable since elimination phases in the concentraation-time profile could not bee identified with certainty. This is also reflected in the e=xtrapolation of AUC data to AUC.

The tissue tao plasma and blood to plasma ratio-s were greater than one for alll time points, suggesting that there was substantial partitionimng of ['“C]-tigecycline-derivexd radioactivity into tissues and blood cells. The tissue tao plasma ratio results from thmis study are similar to tissue= to plasma ratio results from the ra—t following IV dose of minoc=ycline.

While not being bound by theory, the high radioactivity concentrations in tine bone may be due to chelation of tigecycline to calcium. Th e ability of tetracyclines (minocycline, choloretetracyclines) to form chelation complexes with calcium or o ther metal ions and therecby adhere to bone has been described in the literature. In the c urrent study ['*C)-tigecyc dine-derived radioactivity was significantly retained in bone, wish an

AUC of 794704 ng equiv-hr/g. This value is approxirmnately 75-fold greater than pl asma.

An apparent elimin ation half-life of 217 hours was alsso observed in bone. The retention of radioactivity in boone may account for the somewha_t incomplete recovery (89.4 dk

2.50%) of ['“C]-tigecycline in a mass balance study in male Sprague-Dawley rats obsemrved followirag a 5 mg/kg intravenous dose. Exposure C AUC) in bone was 2.5-fold higher than the next highest tissue (thyroid). ['*C]-tigecycline -derived radioactivity also showed a strong a ffinity and long half-life for bone and thyroid tissues which is also similar to other

S$ known tetracyclines. [“CJ-tigecycline-derived radioactivity concentrations were detectable up to 336 hours ime the kidney and were higher than those of the other tissues except for the bone and thyroid. However, in the mass balance study as w ell as biliary and urinary excretion study, most of the ['*C]-tigecycline-derived radioactivity was excreted in the first 48 hours, suggesting that some [**C)-tigecycline-deri-ved radioactivity may be binding with high affinity to the kidney tissue. Binding to kidn ey tissue is also known with tetracyclines. _As determined by QWBAR, radioactivity gpresent in rat ocular tissues was selectiv <ly partitioned only into the melanin-containing tissues of the uveal tract in addition to the skin in the Long-Evans rats. The wveal tract had relatively high concentrations of radioactivity at all time points after 0.5 hours, suggesting a significart level of” exposure and a long half-life. In a previously conducted study using the tissuez dissecti«on method, evaluation of intact eyeball revealed radioactivity was present in th is organ; however, it was not possible to associate thme location of this radioactivity to any specific ocular tissues.

Concentrations of the 14 standards and 28 QCS determined by conventional licquid scintillation counting (LSC) was similar to that of” QWBAR evaluations for these same standarcds. Exposure of these standards to 14 different storage phosphor screens resulteed in a reli able MCID response that correlated with tthe LSC determined specific activitie s, suggesting that intra-day and inter-day variability was very low. The CV and accuracsy of the QW" BAR method were within acceptable limits (< 20%). The reproducibility of thme

MCID wesponse and good correlation of the specific activities between conventional L SC and QWBAR demonstrated that the RBC standards were of uniform concentration of radioactivity. The variability observed in this stucly was considered to be related to various aspects of cryosectioning, QWBAR techn ique and imaging analysis. QWBAR was shown to be reproducible with a sensitivity o£ 0.221 nCi/g (lower limit of quantitation). The dynamic range was linear acro ss four orders of magnitude from 0.2.21 to 832 mCi/g.

In conclusion, tissue concentrations of ['*C]-tigecycline-derived radioactivity were higher for most tissuess compared to plasma concentratio ns.

In general, the elimination of radioactivity from mosst of tissues was slower than the raate from plasma.

AUC wass higher for most tissues that plasma, suggesting that most of the tissues were slow in elimi nating ["“C]-tigecycline-derived radioactivity.

Claims (1)

1. ) PCT/US2004/0228980 CLAIMS:

   i. Use of a pharmaceologically effective amount of tigzecycline for treating bone, bone marrow or joint infections in & mammal.

   2. Use of a pharmacologically effective amount of tigecycline and an antimic robial agent selected from the group consisting of rifamycin, rifampin, rifapentine, rifaxim in, or streptovaricin for treating bore, bone marrow or joint infections in a mammal.

   3. Use of a pharma-cologically effective amount of #&igecycline for manufactures of a medicament for treatment of bone, bone marrow or joint infect® ons in a mammal.

   4. Use of a pharmamcologically effective amount of tigecycline and an antimicrobial agent selected from the gr-oup consisting of rifamycin, rifeampin, rifapentine, rifaxinmin, or streptovaricin for manufactuare of a medicament for treatmerat of bone, bone marrow owr joint infections in a mammal.

   5. The use of any ome of claims 1 - 4, wherein the borne or bone marrow infectior cause osteomyelitis.

   6. The use of any «one of claims 1 - 4, wherein the joint infection or infectiorm of the tissues surrounding the joint= cause septic arthritis.

   7. Use of a pharma cologically effective amount of tigzecycline and rafampin for “treating bone, bone marrow or joint infections in a mammal.

   8. The use of any one of claims 1 - 4 and 7, whe re the infection is comprissed of a pathogen selected from thes group consisting of gram negative bacteria, gram positive bacteria, anaerobic bacteria, and aerobic bacteria. 53 AMENDED SHEET

   PCT/US2004/028980

   9. The use of claim 8 where the pathogen is selecte=d from the group consisting of Staphylococcus, Acinetobacter, Mycobacterium, Haemophilus, Salmonella, Streptococcus, Enterobacteriaceae, Enterococcus, Escherichia, Pseudomonas, Neisseria, Rickettsia, Pneumococci. Prevotella, Peptostreptococci, Bacteroides Legio nella, beta-haemolytic streptococci, group B streptococcus and spirochaetes.

   10. The use of claim 9 wherein the infection is compprised of Neisseria, Mycobacterium, Staphylocoeccus, and Haemophilus.

   1. The use of claim 10 wherein the infection is comprised of Neisseria meningitidis, Mycobacterium tuberculosis, Mycobacterium leprae, Staphylococcus aureus, Staphylococcus epidermidis, or Haemophilu s influenzae.

   12. The use of claim 8 where the pathogen exhibits antibiotic resistance.

   13. The use of clzaim 12 where the antibiotic resistan ce is selected from the group consisting of methicillin res istance, glycopeptide resistance, tet racycline resistance, oxytetracycline resistance, doxycycline resistance, chlortetracy cline resistance, minocycline resistance, glycylcycline resistance, cephalosporin resistance, ciprofloxacin resistance, nitrofurantoin resistance, tri methoprim-sulfa resistance, piperaciilin/tazobactam resistance, moxifloxacin resistance, vancomycin resistance, teicoplanin resistance, penicillin resistance, ancl macrolide resistance.

   14. The use of cLaim | 3 where the glycopeptide resistance is vancomycin resistance.

   15. The use of cLaim 9 where the pathogen is selected from the group consisting of Staphylococcus aureus, Stcaphylococcus epidermidis, Streptococcus pneumoniae, Or Streptococcus pyogenes.

   16. The use of cMaim 15 where the infection is comprised of Staphylococcus aureus.

   17. The use of claim | § where the Staphylococcus caureus exhibits an antibiotic resistance selected from thes group consisting of glycopeptide resistance, tetracycline resistance, minocycline resistance, me=thicilin resistance, vancomycin resi_stance and resistance to a glycylcycline antibiotic other than tigecycline. 54 AMENDED SHEET

   PCT/US2(04/028980

   18. The use of claim 9 where the infection is comprisead of Acinetobacter bai~mannii.

   19. The use of claim 18 where the Acinetobacter bauma=nii exhibits an antibio®tic ressistance selected from the groupp consisting of cephalosporin resistance, ciprofloxacin ressistance, nitrofurantoin resistan_ce, trimethoprim-sulfa resistance, and piperacillin/tazobeactam resistance.

   200. The use of claim 9 where the infection is comprised of Mycobacterium ab-scessus. ’a The use of claim 20) where the Mycobacterium abscezssus exhibits moxifloxacin ressistance.

   22. The use of claim 2 1 where the infection is comprised of Haemophilus inflienzae.

   23. The use of claim 9 where the infection is comprisecd of Enterococcus faecium.

   24. The use of claim 9 where the infection is comprisec of Escherichia coli.

   25. The use of claim 9 where the infection is comprise of Neisseria gonorrizoeae.

   265. The use of claim 9 where the infection is comprisecd of Rickettsia prowazeekii, Rickettsia typhi, or Rickettsia ricksettsii.

   27. Use of a pharmaco logically effective amount of tigeacycline for treating a joint infection or an infection of surroumnding tissues of the joint in a marmmal.

   28. Use of a pharmaco Jogically effective amount of tige<ycline and an antimic Tobial agent selected from the group cormsisting of rifamycin, rifampin, rif apentine, rifaximin, or- stresptovaricin for treating a joint Enfection or an infection of surrounding tissues of the joi nt in a ma_mmal.

   29. The use of claim 28 where the antimicrobial is rifampin.

   30. The use of any one of claims 27-29 where the infec tion is comprised of a pat hogen selected from the group consisting of gram negative bacteria, gram positive bacteria, anamerobic bacteria, and aerobic bamcteria. 55 AMENDED SHEET

   PCT US2004/028980

   31. The use of claim 30 where the pathogen is selected from the group consisting of Staphylococcus, Acinetobacter, Mycobacterium, Haenaophilus, Salmonella, Streptococcus, Enterobacteriaceae, Enterococcus, Escherichia, Pseucdomonas, Neisseria, Rickettsia, Pneumococci, Prevotella, Peptostreptococci, Bacteroiedes Legionella, beta-haemol®ytic streptococci, group B streptococcus and spirochaetes.

   32. The use of claim 31 wherein the infectiomn is comprised of Neisseria, Mycobacterium, Staphylococcus, and Haemophilus.

   33. The use of claim 32 wherein the infectiomn is comprised of Neisseria rneningitidis, Mycobacterium tuberculosis, Mycobacterium leprae, Sstaphylococcus aureus, StapFaylococcus epidermidis, or #4aemophilus influenzae.

   34. The use of claim 30 where the pathogen exhibits antibiotic resistance .

   35. The use of claim 34 where the antibiotic resistance is selected from tle group consisting of me=thicillin resistance, glycopeptide resist. ance, tetracycline resistance. oxytetracycline wresistance, doxycycline resistance, chloOrtetracycline resistance, mimocycline resistance, glycy~lcycline resistance, cephalosporin resistance, ciprofloxacin resistarmce, nitrofurantoin re sistance, trimethoprim-sulfa resistance=, piperacillin/tazobactam res istance, moxifloxacin resistance, vancomycin resistance, teicoplanin resistance, penicillin resistance, and macrolide resistance.

   36. Thee use of claim 35 where the glycopeptmde resistance is vancomycin resistance. : 37. Th_e use of claim 31 where the pathogen is selected from the group co- nsisting of Staphylococcus caureus, Staphylococcus epidermidis, Sereptococcus pneumoniae, or= Streptococcus pyogenes. 38: Th_e use of claim 37 where the infection i=s comprised of Staphylococcus aureus.

   39. Th e use of claim 38 where the Staphylococcus aureus exhibits an anti biotic resistance selected from the group consisting of glycopeeptide resistance, tetracyclinee resistance, minocycline resistance, methicilin resistance, vancomyscin resistance and resistance to a glycylcycline antibiotic other than tigecycline. 56 AMENDED SHEET

   PCT/UNS2004/028980

   40. The use of claim 31 where the infection is co-mprised of Acinetobacter baumannii.

   41. The use of clai-m40 where the Acinetobacter baumanii exhibits an an tibiotic resistance selected from the gzroup consisting of cephalosporin resistance, ciprofloxacin resistance, nitrofurantoin resmstance, trimethoprim-sulfa ressistance, and piperacillin/“tazobactam resistance.

   42. The use of claim 31 where the infection is co=mprised of Mycobacterimum abscessus. 43, The use of clai m 42 where the Mycobacteriu. m abscessus exhibits moxifloxacin resistance. 44, The use of clai_m 31 where the infection is commprised of a pathogen seslected from the group consisting of Haeranophilus influenzae, Enterococ-cus faecium, Escherichica coli, Neisseria gonorrhoeae, Rick=ettsia prowazekii, Rickettsia typhi, or Rickettsia rickett-sii. 45, The use of clai.m 30 wherein the joint infectimon or infection of the sur-rounding tissues of the joint cause septic arthritis.

   46. Use of clairm 1 or claim 2 or claim 7 or claim 27 or claim 28, ssubstantially as herein described with reference to and as illustrated in a_ny of the examples and accompanying drawings.

   47. Use of claim 3 or claim 4, substantially ass herein described with reference to and as illustrated in any of the examples and accompanying d rawings. 57 AMENDED SHEE_T