WO2015179074A1

WO2015179074A1 - Improved antibacterial therapy

Info

Publication number: WO2015179074A1
Application number: PCT/US2015/027876
Authority: WO
Inventors: Mark J. POHL
Original assignee: Health Science Funding Llc
Priority date: 2014-05-19
Filing date: 2015-04-28
Publication date: 2015-11-26

Abstract

The activity of certain antibiotics which function by binding to the r50S ribosomal subunit of bacterial r70s ribosome to inhibit prokaryotic ribosomal translocation, my be increased by certain substrates of a certain subclass of CYP450, i.e., CYP450 oxidases found in vivo in human smooth endoplasmic reticulum. Combining a suitable CYP450 substrate with a suitable antibiotic may thus increase the efficacy of the antibiotic, without increasing the dose and thus avoiding adverse side effects appurtenant to high dose antibiotic therapy.

Description

Improved Antibacterial Therapy

Related Applications

This application claims priority from United States patent application Serial No. 14/281036 filed 13 May 2014, which in turn claims priority from US Serial No. 61/836795, the contents of which are incorporated here by reference.

Government Interest

None.

Background

Macrolide and lincosamide antibiotic compounds impair prokaryotic replication by interfering with prokaryotic protein synthesis. Certain of these antibiotics are metabolized by the cytochrome P450 system. I here propose improving antibacterial therapy by administering a substrate of a CYP450 dehydrogenase because the substrate may by competitive inhibition improve the efficacy of the antibiotic.

Detailed Description

Elongation of the procaryotic polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit.

Procaryotic protein elongation starts when the fJVIet-tRNA enters the 70s ribosome P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor-Tu (EF-Tu), a small GTPase. The P site thus contains the beginning of the peptide chain of the protein to be encoded and the A site has the next amino acid to be added to the peptide chain. The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site. This process, known as peptide bond formation, is catalyzed by a ribozyme (the 23 S ribosomal RNA in the 50S ribosomal subunit). Now, the A site has the newly formed peptide, while the P site has an uncharged tRNA (tRNA with no amino acids). The newly formed peptide in the A site tRNA is known as dipeptide and the whole assembly is called dipeptidyl-tRNA. The tRNA in the P site minus the amino acid is known to be deacylated. In the final stage of prokaryotic polypeptide elongation, called translocation, the deacylated tRNA (in the P site) and the dipeptidyl-tRNA (in the A site) along with its corresponding codons move to the E and P sites, respectively, and a new codon moves into the A site. This process is catalyzed by elongation factor G (EF-G). The deacylated tRNA at the E site is released from the ribosome during the next A-site occupation by an aminoacyl-tRNA again facilitated by EF-Tu. The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).

Erythromycin displays bacteriostatic activity or inhibits growth of bacteria, especially at higher concentrations, but the mechanism is not fully understood. Erythromycin binds to the 50s subunit of the bacterial 70s rRNA complex. By binding to the 50s subunit of the bacterial 70s rRNA complex, protein synthesis and subsequent structure and function processes critical for life or replication are inhibited. Erythromycin interferes with ribosomal translocation, preventing the transfer of the charged tRNA bound at the A site of the rRNA complex to the P site of the rRNA complex. Without this translocation, the A site remains occupied and, thus, the addition of an incoming charged tRNA and its attached amino acid to the nascent polypeptide chain is inhibited. This interferes with the production of functionally useful proteins, which is the basis of this antimicrobial action. Erythromycin is easily inactivated by gastric acid; therefore, all orally-administered formulations are given as either enteric-coated or more-stable salts or esters, such as erythromycin ethylsuccinate. Erythromycin is very rapidly absorbed, and diffuses into most tissues and phagocytes. Due to the high concentration in phagocytes, erythromycin is actively transported to the site of infection, where, during active phagocytosis, large concentrations of erythromycin are released.

Most of erythromycin is metabolised by demethylation in the liver. Its main elimination route is in the bile. There is little renal excretion. Erythromycin's elimination half-life is 1.6 hours. Erythromycin is metabolized by certain enzymes of the cytochrome P450 system, in particular, by isozymes of the CYP3A superfamily, CYP3A.

The activity of the CYP3A enzymes can be induced or inhibited by certain drugs (e.g. dexamethasone) which can cause it to affect the metabolism of many different drugs, e.g. erythromycin. If other CYP3A substrates (drugs that are broken down by CYP3A) such as simvastatin (Zocor), lovastatin (Mevacor), or atorvastatin (Lipitor) are taken concomitantly with erythromycin, levels of the substrates will increase, often causing adverse effects. A noted drug interaction involves erythromycin and simvastatin, resulting in increased simvastatin levels and the potential for rhabdomyo lysis.

Another group of CYP3A4 substrates are drugs used for migraine such as ergotamine and dihydroergotamine; their adverse effects may be more pronounced if erythromycin is associated. Earlier case reports on sudden death prompted a study on a large cohort that confirmed a link between erythromycin, ventricular tachycardia, and sudden cardiac death in patients also taking drugs that prolong the metabolism of erythromycin (like verapamil or diltiazem) by interfering with CYP3A4. Hence, erythromycin should not be administered to people using these drugs, or drugs that also prolong the QT interval. Other examples include terfenadine (Seldane, Seldane- D), astemizole (Hismanal), cisapride (Propulsid, withdrawn in many countries for prolonging the QT time) and pimozide (Orap). Theophylline, which is used mostly in asthma, is also contraindicated.

Clindamycin is a semisynthetic derivative of lincomycin, a natural antibiotic produced by the actinobacterium Streptomyces lincolnensis. It is obtained by 7(5)-chloro-substitution of the 7(i?)-hydroxyl group of lincomycin. The synthesis of clindamycin was first announced by BJ Magerlein, RD Birkenmeyer, and F Kagan on the fifth Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in 1966. It has been on the market since 1968.

Clindamycin has a bacteriostatic effect. It is a bacterial protein synthesis inhibitor by inhibiting ribosomal translocation, in a similar way to macrolides. It does so by binding to the 50S rRNA of the large bacterial ribosome subunit. Clindamycin may prolong the effects of neuromuscular-blocking drugs, such as succinylcholine and vecuronium. Its similarity to the mechanism of action of macrolides and chloramphenicol means they should not be given simultaneously, as this causes antagonism and possible cross-resistance.

Macrolide antibiotics such as clarithromycin inhibit the metabolic pathway of statins via the cytochrome CYP450 3A4 hepatic enzyme system and may result in elevated CK level, myopathy, or rhabdomyolysis

Erythromycin is not recommended when using clindamycin-containing products, even topical products such as Duac® or BenzaClin®. In general, the simultaneous use of two different erythromycin derivatives (such as clindamycin and Mitemcinal) should be avoided as drugs in this macrolide family possess a common mechanism of action. The inhibition of CYP2D6 and CYP3A4 enzyme activity with telithromycin affects the pharmacokinetics and pharmacodynamics of orally administered oxycodone. In a randomized 2- phase crossover study, eleven (11) healthy subjects were pretreated with 800 mg of oral telithromycin or placebo for 4 days. On day 3, they ingested 10 mg of immediate -release oxycodone. Plasma concentrations of oxycodone and its oxidative metabolites were measured for 48 hours, and pharmacodynamic effects were evaluated. Telithromycin increased the area under the plasma concentration-time curve (AUC(O-infinity)) of oxycodone by 80% (P < .001) and reduced the AUC(O-infinity) of noroxycodone by 46% (P < .001). Most of the pharmacokinetic changes were seen in the elimination phase, with little effect by telithromycin on the peak concentration of oxycodone. Pharmacodynamic effects of oxycodone were modestly enhanced by telithromycin. This study showed that telithromycin reduces N-demethylation of oxycodone to nor-oxycodone by inhibiting CYP450 3A4.

Similarly, aluminum is a mildly toxic metal and can accumulate in the liver. The hepatic microsomal cytochrome P450 enzyme system (CYPS) plays important role in the transformation of such toxic materials. In a study to investigate the effects of aluminum trichloride (A1C1₃) on the rat CYPS, forty male Wistar rats (5weeks old) weighing 110-120g were randomly allocated and orally exposed to 0, 64.18, 128.36 and 256.72mg/kg body weight (BW) AICI₃ in drinking water for 120 days. The body weight (BW) of rats, hepatosomatic index (HSI), hepatic Al content, the concentrations of cytochrome P450 (CYP450), cytochrome B5 (B5), microsomal protein and the activities of NADPH-cytochrome c reductase (CR), aminopyrin N-demethylase (AND), erythromycin N-demethylase (ERND) and aniline-4-hydeoxylase (AH) were assessed at the end of the experiment. The results showed that increased aluminum decreased BW, HIS, concentrations of CYP450, B5, microsomal protein and the activity of CR, AND, ERND and AH in hepatic microsomes. The results revealed that exposure to AICI₃ inhibited the microsomal CYP450 dependent enzyme system of liver.

Similarly, ASAHl suppression increases the transcription of multiple steroidogenic genes, including Cytochrome P450 monooxygenase (CYP)17A1, CYPl lBl/2, CYP21A2, steroidogenic acute regulatory protein, hormone-sensitive lipase, 18-kDa translocator protein, and the melanocortin-2 receptor. In H295R human adrenocortical cells, ACTH rapidly activates ceramide (Cer) and sphingosine (SPH) turnover with a concomitant increase in SPH-1 -phosphate secretion. These bioactive lipids modulate adrenocortical steroidogenesis, primarily by acting as second messengers in the protein kinase A/cAMP-dependent pathway. Acid ceramidase (ASAHl) directly regulates the intracellular balance of Cer, SPH, and SPH-1 -phosphate by catalyzing the hydrolysis of Cer into SPH. ACTH/cAMP signaling stimulates ASAHl transcription and activity, supporting a role for this enzyme in glucocorticoid production. The role of ASAHl in regulating steroidogenic capacity was examined using a tetracycline-inducible ASAHl short hairpin RNA H295R human adrenocortical stable cell line. The data show that ASAHl suppression increases the transcription of multiple steroidogenic genes, including Cytochrome P450 monooxygenase (CYP)17A1, CYP11B1/2, CYP21A2, steroidogenic acute regulatory protein, hormone-sensitive lipase, 18-kDa translocator protein, and the melanocortin-2 receptor. Induced gene expression positively correlated with enhanced histone H3 acetylation at target promoters. Repression of ASAHl expression also induced the expression of members of the nuclear receptor nuclear receptor subfamily 4 (NR4A) family while concomitantly suppressing the expression of dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1. ASAHl knockdown altered the expression of genes involved in sphingo lipid metabolism and changed the cellular amounts of distinct sphingo lipid species. Perhaps most pertinently for the instant claimed invention, ASAHl silencing increased basal and cAMP-dependent Cortisol and dehydroepiandrosterone secretion, establishing ASAHl as a pivotal regulator of steroidogenic capacity in the human adrenal cortex.

Similarly, roxithromycin and its metabolites showed weak inhibitory effects on CYP450. Liver microsomes of Wistar rats, induced by phenobarbital, were prepared using ultracentrifuge method. RXM in vitro metabolism was studied with the microsome incubation. The metabolites were separated and assayed by liquid chromatography-tandem mass spectrometry (LC-MSn). N- Mono- and N-di-demethyl metabolites as well as O-dealkylated metabolite (erythromycin oxime) were detected in microsomal incubates. Roxithromycin and its metabolites expressed weak potency to form inactive complexes with CYP450. N-Demethylation and oxime ether side chain O- dealkylation are main biotransformation pathways of roxithromycin in phenobarbital-treated rat liver microsomes. Both routes were found to be NADPH-dependent. Roxithromycin and its metabolites thus were confirmed to have (weak) inhibitory effects on CYP450.

The antibiotics erythromycin and clarithromycin are potent inhibitors of CYP3A4 and can increase blood levels and toxicity of CYP3A4 substrates. Likewise, quinolone antibiotics such as ciprofloxacin inhibit the metabolism of CYP1A2 substrates. Other dental therapeutic agents are substrates for CYP2C9 (celecoxib, ibuprofen and naproxen), CYP2D6 (codeine and tramadol), CYP3A4 (methylprednisolone) and CYP2E1 (acetaminophen). Because codeine and tramadol are prodrugs, inhibition of their metabolism can lead to a diminution of their analgesic effects. While inducers of acetaminophen metabolism, including alcohol, theoretically can increase the proportion of it that is biotransformed into a potentially hepatotoxic metabolite, recent research suggests that concomitant alcohol intake does not increase the hepatotoxic potential of therapeutic doses of acetaminophen. A number of clinically significant drug interactions can arise with dental therapeutic agents that act as substrates or inhibitors of the CYP450 system.

Effects of erythromycin on hepatic CYP450 3A4 isozymes can profoundly influence the metabolism of many therapeutic agents. An open-label, randomized, two-period, crossover study was therefore conducted to evaluate the pharmacokinetics of felbamate before and after a concurrent 10-day regimen (333 mg three times daily) of erythromycin. Patients were receiving either 3,000 or 3,600 mg/day felbamate monotherapy for treatment of epilepsy. Mean dose- normalized values for maximum concentration (Cmax) and area under the concentration-time curve (AUC tau) of felbamate were not statistically different in patients taking felbamate as monotherapy than in patients after erythromycin coadministration. Estimates of time to Cmax (tmax), minimum concentration (Cmin), apparent clearance (Cl/kg), average concentration (Cav), and degree of fluctuation (DFss) were likewise unchanged. The incidence of mild and moderate adverse events increased during coadministration of the two drugs. Because patients with epilepsy can not be treated with erythromycin alone, it could not be determined whether the adverse events were attributable to erythromycin or to the combination of the two drugs. Steady- state pharmacokinetic parameters of felbamate were not influenced by erythromycin coadministration.

P-glycoprotein (P-gp), multiple drug resistance associated proteins (MRPs), and cytochrome P450 3A4 together constitute a highly efficient barrier for many orally absorbed drugs. Multidrug regimens and corresponding drug-drug interactions are known to cause many adverse drug reactions and treatment failures. Available literature, clinical reports, and in vitro studies indicate that many drugs are substrates for both P-gp and CYP3A4. MDCKII-MDR1 was employed as an in vitro model to evaluate the effects of antiretrovirals, azole antifungals, macrolide, and fluroquinolone antibiotics on efflux transporters. Ketoconazole (50 muM) enhanced the intracellular concentration of (3)H ritonavir. The inhibitory effects of ketoconazole and MK 571 on the efflux of (3)H ritonavir were comparable. An additive effect was observed with simultaneous incorporation of ketoconazole and MK 571. Results of (3)H ritonavir uptake studies were confirmed with transcellular transport studies. Several fluroquinolones were also evaluated on P-gp-mediated efflux of (3)H cyclosporin and 14C erythromycin. These in vitro studies indicate that grepafloxacin, levofloxacin, and sparfloxacin are potent inhibitors of P-gp- mediated efflux of 14C erythromycin and (3)H cyclosporin. Those data show that simultaneous administration of fluoroquinolones and macrolides could minimize the efflux and metabolism of both drugs.

Similarly, the effects of erythromycin and ketoconazole on carbamazepine metabolism are known to effect formation of 10,11-epoxy carbamazepine, a major CBZ metabolite, which is significantly inhibited by these agents.

This background leads to the instant use of certain CYP substrates in conjunction with certain prokaryotic ribosomal translocation inhibitors. Suitable prokaryotic ribosomal translocation inhibitors include erythromycin, its derivative clindamycin, and similar compounds. Preferably, the prokaryotic ribosomal translocation inhibitor is provided in a strength of from about 0.5% to 3%. It may optionally be combined with benzoyl peroxide or a similar agent. Optionally, the prokaryotic ribosomal translocation inhibitor may be formulated as a topical dosage form.

Similarly, the CYP substrate may be formulated as e.g., a topical or oral dosage form. The substrate must be a substrate of at least one of the species of cytochrome P450 dehydrogenase which are normally found in the smooth endoplasmic reticulum of human cells. Suitable substrate thus includes, for example, substrates of 3-beta-hydroxysteroid dehydrogenase and / or 17-beta- hydroxysteroid dehydrogenase. To avoid precipitating unwanted ancillary biological effect, the substrate is preferably not hormonally active. The substrate may also be a naturally-occurring compound. The most preferred example of substrate is 5- dehydroepiandrosterone, a compound which is normally secreted by the adrenal cortex, is itself apparently inert, and which is in local tissues converted as and where needed in vivo by CYP450 into biologically-active compounds. The substrate dose is preferably an amount which will competitively bind to the CYP 450 dehydrogenase in an amount sufficient to competitively reduce the availability of that CYP 450 enzyme to react with other compounds. The dosage of 5- dehydroepiandrosterone is expected to be between about 25 and about 250 mg / day for a normal adult human; other substrates will have different dosages required depending on their affinity for the CYP 450 enzyme. Enzyme affinity and resulting dosage may be calculated using the high throughput screening (HTS) method described in Doshi et al. (2011), which reports assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIP A). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1- aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96- well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metabolism, whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. Doshi says that the high sensitivity and specificity of their assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large number of chemicals in the drug discovery phase for CYP3A4 inhibitory and inducing potential.

Given this general disclosure, the skilled artisan could readily derive modifications to it. Thus, I intend the legal coverage of my patent to be defined not by the specific examples described here, but by the legal claims the Patent Office has reviewed and approved.

Claims

I claim:

1. In a method of administering to a patient a pharmaceutical finished dosage form of an antibiotic compound which interferes with prokaryotic r50S ribosomal translocation, the improvement comprising:

administering a second compound, said second compound characterized in that it is a substrate of a cytochrome P450 dehydrogenase enzyme normally found in the smooth endoplasmic reticulum of human cells,

wherein the cytochrome P450 dehydrogenase enzyme normally found in the smooth endoplasmic reticulum of human cells is selected from the group consisting of: 3-beta-hydroxysteroid dehydrogenase and 17-beta- hydroxysteroid dehydrogenase,

said antibiotic compound provided in an antibiotic effective amount, and

said second compound provided in an amount effective to competitively bind to said cytochrome P450 dehydrogenase enzyme.

2. The method of Claim 1 above, wherein the antibiotic compound which interferes with prokaryotic r50S ribosomal translocation is selected from the group consisting of: erythromycin and clindamycin.

3. The method of Claim 2 above, wherein the antibiotic pharmaceutical finished dosage form is a topical pharmaceutical finished dosage form comprising from about 0.5% to about 3.5% (w/w) of the antibiotic.

4. The method of Claim 3 above, wherein the topical pharmaceutical finished dosage form further comprises benzoyl peroxide.

5. The method of Claim 1 above, wherein the second compound is selected from the group consisting of: androstenediol and 5-dehydroepiandrosterone.

6. The method of Claim 5 above, wherein the second compound is provided in a topical finished dosage form.

7. The method of Claim 5 above, wherein the second compound is provided in an oral finished dosage form.

8. The method of Claim 7 above, wherein said amount of said second compound effective to competitively bind to said cytochrome P450 dehydrogenase enzyme comprises an oral dose of from about 25 to about 250 mg of said second compound.

9. The method of claim 5, wherein the second compound comprises an oral dose of from about 25 to about 250 mg of androstenediol.

10. The method of claim 5, wherein the second compound comprises an oral dose of from about 25 to about 250 mg of 5-dehydroepiandrosterone.

11. A kit containing:

the pharmaceutical finished dosage form of an antibiotic compound which interferes with prokaryotic r50S ribosomal translocation of Claim 1, and

the a substrate of a cytochrome P450 dehydrogenase enzyme of Claim 1,

said antibiotic compound provided in an antibiotic effective amount, and

said substrate of a cytochrome P450 dehydrogenase enzyme provided in an amount effective to competitively bind to said cytochrome P450 dehydrogenase enzyme.

12. The kit of Claim 11 above, wherein the antibiotic compound which interferes with prokaryotic r50S ribosomal translocation is selected from the group consisting of: erythromycin and clindamycin.

13. The kit of Claim 12 above, wherein the antibiotic pharmaceutical finished dosage form is a topical pharmaceutical finished dosage form comprising from about 0.5% to about 3.5% (w/w) of the antibiotic.

14. The kit of Claim 13 above, wherein the topical pharmaceutical finished dosage form further comprises benzoyl peroxide.

15. The kit of Claim 11 above, wherein the second compound is selected from the group consisting of: androstenediol and 5-dehydroepiandrosterone.

16. The kit of Claim 15 above, wherein the second compound is provided in a topical finished dosage form.

17. The kit of Claim 15 above, wherein the second compound is provided in an oral finished dosage form.

18. The kit of Claim 17 above, wherein said amount of said second compound effective to competitively bind to said cytochrome P450 dehydrogenase enzyme comprises an oral dose of from about 25 to about 250 mg of said second compound.

19. The kit of claim 15, wherein the second compound comprises an oral dose of from about 25 to about 250 mg of androstenediol.

20. The kit of claim 15, wherein the second compound comprises an oral dose of from about 25 to about 250 mg of 5-dehydroepiandrosterone.