PHARMACEUTICAL COMPOSITION COMPRISING A DRUG CONTAINING AT LEAST ONE TOXICOPHORE FUNCTION AND N-ACETYL-L- CYSTEINE
DESCRIPTION
The present invention relates to a pharmaceutical composition containing at least one drug containing at least a toxicophore function, N-acetyl-L-cysteine (NAC), at least one antioxidant agent and at least one pharmaceutically acceptable excipient, and the same for use in the prevention and/or reduction of the occurrence of idiosyncratic adverse drug reactions ( IADRs ) caused by said drug.
Adverse drug reactions (ADRs) can be classified as predictable (type A reactions) or idiosyncratic (type B reactions ) .
Type A reactions are dose-dependent and occur in a relatively consistent time frame; all individuals are susceptible. A typical example is acetaminophen induced hepatotoxicity (Larson AM, Poison J, et al . Acute Liver Failure Study Group. Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology. 2005 Dec ; 42 ( 6 ): 1364-72 ; Amar PJ, Schiff ER. Acetaminophen safety and hepatotoxicity-where do we go from here? Expert Opin Drug Saf. 2007 Jul ; 6 ( 4 ) : 341-55. ) .
One cause of drug toxicity is related to the formation of reactive species generated by the drug itself. The reactive species can bind with proteins or enzymes leading to cell apoptosis, necrosis, perturbation of the host immuno system or allergies, or with DNA leading to mutagenicity or carcinogenicity.
Glutathione (GSH) is an antioxidant produced within the body that is involved in detoxification processes, since it forms stable and water-soluble adducts with the reactive species originated by some drugs, removing them from circulation.
GSH levels than constitute an important threshold that determines the toxicity of a drug able to form reactive intermediates species, as it determines the occurrence of the dose-dependent predictable adverse drug reactions (ADRs, type A reactions).
GSH depletion may originate from the administration of high doses of drugs, due to the fact that GSH binds to the high amounts of reactive intermediate metabolites released by said drugs .
It follows that GSH levels are mostly relevant for acute toxicit .
Unlike the adverse drug reactions of type A, idiosyncratic adverse drug reactions ( IADRs ) occur in a minority of patients during drug therapy and are either unrelated to the pharmacological action of the drug or to the dose of the administered drug (Senior JR. What is idiosyncratic hepatotoxicity? What is it not? Hepatology. 2008 Jun ; 47 ( 6 ) : 1813-5 )
IADRs occurrence is not directly related to the administered dose of a drug, however it is obvious that higher doses, by generating higher amounts of reactive metabolites, increase the probability of IADRs. The final response is indeed the product of a number of factors: drug dose, amount and reactivity of electrophile metabolites, patient physiological condition and reactivity of the immunological system.
IADRs are currently unpredictable and difficult to diagnose, and they occur at doses that do not normally cause toxicity. They typically display a variable onset time after the beginning of drug therapy and they may also occur in a later stage, far-off from the drug administration ( aplowitz N. Idiosyncratic drug hepatotoxicity . Nat Rev Drug Discov. 2005 Jun; 4 ( 6 ) : 489- 99. Review; Waring JF, Anderson MG . Idiosyncratic toxicity: mechanistic insights gained from analysis of prior compounds. Curr Opin Drug Discov Devel. 2005 Jan ; 8 ( 1 ) : 59-65. Review; Uetrecht J. Idiosyncratic drug reactions: past, present, and future. Chem Res Toxicol. 2008 , Jan;21 ( 1 ) : 84-92. Epub 2007 Dec 4. Review; Williams DP, Park BK. Idiosyncratic toxicity: the role of toxicophores and bioactivation . Drug Discov Today. 2003 Nov 15;8 (22 ) : 1044-50. Review).
Clinically apparent consequences of IADRs are, for example, hepato-toxicity , skin reactions and blood dyscrasias (Zhang X. et al. Drug Metab. Pharmacokinet . (2011), 26 (1): 47-59).
Differently from Type A reactions, IADRs are difficult to understand and to predict by use of current preclinical testing paradigms or during phase I clinical trials. Better prediction of idiosyncratic drug induced liver injury will require an understanding of the modes and mechanisms of the reactions.
The formation of reactive species originated by some drugs, in conjunction with a decreased ability for GSH detoxification process is believed to be the initial step in many IADRs.
Said reactive species originate from drugs containing the so called toxicophores, i.e. metabolically
activated functions ( enzymatically catalyzed) within a chemical structure. Examples of reactive species are intermediate electrophile metabolites such as quinones, quinone-imine , quinine-methide derivatives.
It seems that once a drug containing a toxicophore forms reactive metabolites, the threshold level to induce IADRs is achieved when a series of concomitant prerequisites is present, such as:
1. Amount and chemical reactivity of the electrophile metabolites high enough.
2. Binding of reactive metabolites to a certain protein with a high epitope density to express an immunogenic potential (hapten specific response).
3. Presence of tissue injuries (cell stress with glutathione depletion) by virus, bacteria, reactive metabolites, inducing up-regulation of co-stimulatory signals for T-cell activation.
4. Failure to down regulating the immuno system.
Mitochondrial damage has been proven to be an important component of the mechanism of idiosyncratic drug- induced liver injury, which can lead to the death of hepatocytes. Screening for mitochondrial functionality is thus well recognized in the art for the evaluation of the safety of drug candidates (Zhang X. et al. Drug Metab. Pharmacokinet (2011), 26 (1): 47-59).
In many cases, IADRs subsides after cessation of treatment, thus posing a significant diagnostic hurdle. In view of their unpredictability, IADRs are up to now contrasted only by suspending the drug administration or by symptoms relief.
N-acetyl-L-cysteine (NAC) is a pharmaceutical drug commonly used as a mucolytic agent (CAS N° 616-91-1).
NAC is also known for its antioxidant properties and liver protecting effects, since it is a precursor of GSH and acts to augment the glutathione reserves in the body: together with glutathione, it directly binds to toxic metabolites.
NAC is therefore currently known, and actually used, for the treatment of hepatic failure of different etiology, particularly in case of acute toxicity due to abuse or overdose of drugs resulting in marked glutathione depletion. NAC is thus normally administered, as an antidote, after the assumption of a drug and it is administered orally or by intravenous infusion in very high dosages. As an example, intravenous NAC is indicated for the treatment of acetaminophen overdose. When taken in large quantities, the toxic benzoquinone imine acetaminophen metabolite accumulates, and the body glutathione reserves are not sufficient to inactivate it. This metabolite is then free to react with key hepatic enzymes, therefore damaging hepatocytes. This may lead to severe liver damage and even death by fulminating liver failure (Fontana, Med Clin N Am 92, (2008), 761-794).
For the treatment of acute acetaminophen poisoning the recommended dosing for NAC is a 150 mg/kg loading dose followed by maintenance doses of 50 mg/kg every 4 hours (Saito et al, Hepathology 2010, Vol 51, 246-254).
It is therefore clear that taking into account the normal weigh of a subject the above dosages per kilogram (mg/kg) represent a very high dosage.
N-acetyl-L-cysteine (NAC) has never been used for the prevention of potential idiosyncratic adverse drug reactions ( IADRs ) .
NAC is moreover known to be unstable when formulated for pharmaceutical uses and there is, therefore, the need of a pharmaceutical formulation in which it is formulated with an improved stability.
Bulk of NAC need to be stored in cool rooms, humidity must be avoided (hygroscopic), while contaminant oxidizers and heavy metals must be removed.
Drugs containing toxicophores , giving rise to reactive quinones, quinine-imine , quinine-methide derivatives cause, besides liver ISDRs, skin ISDRs, like macupapular rashes, urticaria, Steven-Johnson syndrome and toxic epidermal necrolysis and include widely used drugs such as atorvastatin , diclofenamic acid or a salt thereof, nimesulide, trazodone, amiodarone, buspirone, carbamazepine , flutamide, minocycline, phenytoin, paroxetine, tacrine, tadalafil, tamoxifen, trimethoprim, nevirapine, trovafloxacin or a mixture thereof .
Atorvastatin is a lipid-lowering agent, able to inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, which catalyzes the conversion of HMG-CoA to mevalonate. Atorvastatin is marketed as its calcium salt. Atorvastatin metabolic pathways give rise to orto and para-hydroxy acetanilido metabolites which can be oxidized to form electrophile quinone imine intermediates that can be trapped by GSH (see scheme 1)( Jacobsen , Kuhn B, Soldner A, Kirchner G, Sewing KF, Kollman PA, Benet LZ, Christians U. Lactonization is the critical first step in the disposition of the 3- hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin. Drug Metab Dispos. 2000 Nov; 28 ( 11 ): 1369-
Atorvastatin calcium tablets available on the market for oral administration contain 10, 20, 40 or 80 mg of active principle.
Scheme 1
Nimesulide, N- ( 4-Nitro-2-phenoxyphenyl ) methane sulfonamide, is a non-selective non-steroidal antiinflammatory drug (NSAID). It is used to treat acute pain, and the symptoms of painful osteoarthritis.
When first put on the market, nimesulide was used to treat a wide range of painful conditions, but concerns
arising over the rare but severe idiosyncratic hepatotoxicity led to restrict its use to the maximum daily dose of 200 mg (Questions and answer on the CHMP recommendation on nimesulide containing medicines London, 21 September 2007. Doc. Ref. EMEA/430988/2007 ) . The mechanisms associated with nimesulide toxicity remain unknown. Nimesulide is rapidly absorbed and it is normally bio-transformed in the liver to metabolites which are then eliminated in the urine/feces ( Bernareggi A. Clinical pharmaco-kinetics of nimesulide. Clin.Pharmacokinet . 1998; 35: 247-274). Two di-imino quinone intermediate metabolites have been detected (see scheme 2) and their electrophilic reactivity, even towards GSH, has been demonstrated by selectively alkylating human serum albumin (HSA) at the free thiol, Cys-34 (Fengping Li et al. In Vitro nimesulide Studies toward Understanding Idiosyncratic Hepatotoxicity: diiminoquinone formation and conjugation; Chem. Res. Toxicol., 22, 72-80, 2009).
Nimesulide is contained in several oral formulations as : immediate release tablets (25, 50 and 100 mg), sachets (100 mg) and syrups (50 mg/5ml).
Scheme 2
Diclofenamic acid or a salt thereof is a worldwide prescribed NSAID used in acute, short-term and chronic treatment regimens.
Although diclofenamate-associated hepatitis was thought to be rare, there are indications that borderline increases in serum transaminases occur in approximately 15% of patients under chronic therapy. Despite some cases report that the adverse hepatic effects are compatible with hypersensitivity reactions, the occurrence of IADRs in other patients appears more consistent with a direct toxic effect of the drug or a drug metabolite ( s ) .
The mechanism by which diclofenamic acid or its salts causes liver alterations in certain individuals is not yet understood, and both the formation of a toxic metabolite and covalent binding of the drug to hepatic proteins have been invoked to explain its toxicity. Recently, the formation of N, 5-dihydroxy-diclofenamate and of the electrophile diclofenac-2 , 5-quinone-imine derivative in activated neutrophils, which could be related to drug toxicity, has been reported (see scheme 3) (Sort R, Ponsoda X, Jover R, Gomez-Lechon MJ, Castell JV. Diclofenac toxicity to hepatocytes : a role for drug metabolism in cell toxicity., J Pharmacol Exp Ther. 1999 Jan; 288 ( 1 ) : 65-72 ) .
Diclofenamic acid, as its sodium (Diclofenac Na) , potassium, diethylamine or epolamine salt, is contained in several oral formulations as immediate release tablets (25, 50 and 75 mg), delayed release tablets (100 mg) sachets (25, 50 mg) and syrups (50 mg/5ml). Injectable formulation are also available (75 mg/3ml)
Scheme 3
Trazodone is an antidepressant chemically unrelated to tricyclic, tetracyclic, or other known anti-depressant agents. There are several cases of IADRs including acute and chronic hepatitis associated with trazodone use. Although the cause of this toxicity is unknown, in most cases liver biopsy specimens from dead patients revealed centrozonal necrosis consistent with a toxic aetiology initiated by electrophile imino quinone or epoxide metabolites (scheme 4) (Kalgutkar AS, Henne KR, Lame ME, Vaz AD, Collin C, Soglia JR, Zhao SX, Hop CE . Metabolic activation of the nontricyclic antidepressant trazodone to electrophile quinone-imine and epoxide intermediates in human liver microsomes and recombinant P4503A4. Chem Biol Interact. 2005 Jun 30 ; 155 ( 1-2 ): 10-20. Epub 2005 Apr 18) .
Trazodone is supplied as trazodone HC1 for oral administration in 50 mg, 100 mg, 150 mg, and 300 mg tablets .
Scheme 4
It has been surprisingly found that the simultaneous administration, in a single pharmaceutical form, of a drug containing at least one toxicophore function with N-acetyl-L-cysteine (NAC), at least one antioxidant agent and at least one physiologically acceptable excipient can prevent and/or reduce the occurrence of idiosyncratic adverse drug reactions ( IADRs ) correlated to said drugs, by preventively avoiding the accumulation of toxic reactive intermediates.
For the purpose of the invention the term "drug" refers to "active ingredient" and, therefore, the term "drug containing at least one toxicophore function" refers to active ingredient containing at least one toxicophore function .
The term "toxicophore function" of the invention then refers to a feature or group within a chemical structure that is thought to be responsible for the toxic properties, either directly or thereby metabolic activation originating reactive species. Examples of reactive species are intermediate electrophile metabolites such as quinones, quinone-imine , quinine- methide derivatives .
The administration of NAC in association with drug(s) containing at least one toxicophore function represents a significant improvement and/or a useful alternative to the GSH detoxification system, while concurrently contributing to maintain the thiol status, wherein thiol status of the invention refers to the pool of SH- containing molecules maintaining the red-ox status, and providing cysteine as a precursor for GSH synthesis. The administration of NAC in association with drugs containing at least one toxicophore function can be therefore usefully used to prevent and/or reduce the occurrence of IADRs in all cases in which the GSH detoxification system is depleted becoming less effective .
The presence of at least one antioxidant agent allows to obtain an improved stability of the N-acetyl-L-cysteine by removing contaminant oxidizers and/or heavy metals possibly present.
Thus, object of the present invention is a pharmaceutical composition comprising at least one drug containing at least one toxicophore function, N-acetyl- L-cysteine, at least one antioxidant agent and at least one physiologically acceptable excipient.
Further object of the present invention is a pharmaceutical composition comprising at least one drug containing at least one toxicophore function, N-acetyl- L-cysteine, at least one antioxidant agent and at least one physiologically acceptable excipient for use in the prevention and/or reduction of the occurrence of IADRs caused by said drug, while maintaining the therapeutic profile of the drug itself.
The pharmaceutical composition of the invention is preferably for use in the prevention and/or reduction of the occurrence of idiosyncratic adverse drug reaction in patients undergoing a treatment with at least one drug containing at least one toxicophore function.
Further object of the present invention is a method for preventing and/or reducing the occurrence of idiosyncratic adverse drug reactions caused by a drug containing at least one toxicophore function which comprises the simultaneous administration, in a single pharmaceutical form, of at least one drug containing at least one toxicophore function, N-acetyl-L-cysteine , at least one antioxidant agent and at least one physiologically acceptable excipient.
According to the invention said idiosyncratic adverse drug reactions are preferably skin diseases (as, for example, macupapular rashes, urticaria, Steven-Johnson syndrome, toxic epidermal necrolysis), liver diseases and/or blood dyscrasias.
According to the present invention, the pharmaceutical composition is preferably administered to mammals, more preferably humans comprising adults and paediatric population wherein the term "paediatric population" refers to subject from born to 18 years old.
According to the invention the N-acetyl-L-cysteine (NAC) is contained in an amount from 1 to 10 fold higher with respect to the drug, on a molar basis.
Preferred drugs of the present invention are those including in their structure at least one toxicophore function, able to release reactive intermediate metabolites such as, for example, the electrophile quinones, imino-quinones , quinone-methides , derivatives .
According to the present invention, the drug containing at least one toxicophore function is preferably selected from atorvastatin , diclofenamic acid or salts thereof (sodium, potassium, diethylamine or epolamine salt), nimesulide, trazodone, amiodarone, buspirone, carbamazepine , flutamide, minocycline, phenytoin, paroxetine, tacrine, tadalafil, tamoxifen, trimethoprim, trovafloxacin and nevirapine. More preferably, the drug is nimesulide or at least one diclofenamic acid salt. Much more preferably, the drug is nimesulide or diclofenamic sodium salt (Diclofenac Na) .
According to preferred embodiments of the invention, the pharmaceutical composition contains nimesulide, diclofenamic acid sodium salt or both.
According to the present invention, said at least one antioxidant is preferably selected from of vitamin E, tioctic acid, sodium metabisulphite and a mixture thereof .
According to the present invention, said at least one antioxidant is present in an amount ranging from 2 mg to 100 mg by weight, based on the total weight of the composition, preferably from 5 mg to 50 mg by weight,
more preferably ranging from 3 to 10 mg by weight for vitamin E, ranging from 10 to 50 mg by weight for tioctic acid and/or ranging from 2 to 5 mg by weight for sodium metabisulphite based on the total weight of the composition. With the aim of further improving NAC stability a chelating agent is added to the composition of the invention, preferably the chelating agent is comprised in an amount ranging from 5 to 20 mg by weight, based on the total weight of the composition and more preferably it corresponds to Na edetate.
The pharmaceutical composition according to the invention is preferably formulated in oral, sub-lingual, or parenteral form; more preferably the pharmaceutical composition of the invention is then formulated in tablet, capsule, sachet, dragee, bead, granule, solution or suspension; much more preferably in tablet.
According to the invention the term "tablet" refers to any kind of tablet comprising single layered(conventional tablet), multi-layered, coated- tablet, minitablet or matrix tablet; the multi-layered tablet of the invention can be preferably formulated as a tab-in tab, bilayered or trilayered tablet in view of the technical effect and/or advantage to be reached, and/or of the selected drug and the layers are preferably isolated one from another.
The "tablet" according to the invention can be preferably obtained by direct compression and/or by wet granulation .
The "multi-layered" according to the invention refers to a tablet comprising at least one layer containing:
N-acetyl-L-cysteine ,
at least one antioxidant, and
optionally at least one physiologically acceptable excipient;
and at least a second layer containing:
at least one drug containing at least one toxicophore function, and
at least one physiologically acceptable excipient.
According to a preferred embodiment of the invention, the term "tab-in-tab" refers to a tablet wherein a smaller tablet is contained, and it is intended to obtain an improved stability of the final tablet and/or of the used drug(s). According to the tab-in-tab embodiment the N-acetyl-L-cysteine and at least one antioxidant optionally with at least one physiologically acceptable excipient are preferably contained in the outer tablet, while at least one of the above listed drugs is preferably contained in the inner tablet with at least one physiologically acceptable excipient.
According to further embodiments the "bilayered tablet" refers to a tablet comprising two layers, superimposed, one containing the N-acetyl-L-cysteine and at least one antioxidant and optionally at least one physiologically acceptable excipient, and the other containing at least one of the above listed drugs and at least one physiologically acceptable excipient; preferably, the by-layered tablet of the invention is directed to a composition in which both layers are for immediate release .
The "trilayered tablet" is then preferably directed to a composition containing three layers: a first layer containing N-acetyl-L-cysteine and at least one antioxidant and optionally at least one physiologically acceptable excipient, an intermediate second, inert,
layer, and a third layer containing at least one of the above listed drugs and at least one physiologically acceptable excipient; the trilayered structure according to the invention is preferably intended for incompatibility of components and/or for combining different release profiles.
The term "inert layer" refers to a layer in which only physiologically acceptable excipients are contained.
According to the invention the above at least one physiologically acceptable excipient can be selected from diluent agents, disintegrant agents, glidant agents, fillers, binding agents, solubilising agents, lubricant agents, release agents, plasticizer agents, chelating/anticoagulant agents, protecting coating agents, preservatives, aromatizer agents and a mixture thereof .
According to the invention suitable disintegrant agents can be preferably selected from crosscaramellose , crosspovidone , cellulose microcrystalline , sodium lauryl sulphate, sodium starch glycolate and a mixture thereof; suitable glidant agents can be preferably selected from corn starch, talc, magnesium trisilicate, colloidal silica anhydrous and a mixture thereof; suitable diluent agents can be preferably selected from povidoneK30/ gelucine, cellulose microcrystalline, lactose, mannitol, sorbitol, kaolin, sucrose, calcium phosphate, threalose, xylitol and corn starch; suitable lubricant agent can be preferably selected from talc, sodium benzoate, poloxamer and a mixture thereof; suitable release agent can be preferably selected from magnesium stearate, sodium stearil fumarate, Ca/Zn stearate, polyethylene glycols (>6000); suitable plasticizer agent can be
preferably selected from polysorbates , glyceril monostearate , triethylcitrate and a mixture thereof; suitable aromatizer agent can be preferably selected from orange powder, citric acid anhydrous or a mixture thereof; suitable chelating agent/anticoagulant is preferably sodium edetate; suitable preservative is preferable benzyl alcohol.
Any other physiologically acceptable excipient can be used for the pharmaceutical composition of the invention .
According to the invention, at least one solvent and/or diluent can be further added to the composition as, for example, water in case of injectable compositions.
The composition of the invention is thus preferably directed to oral, sub-lingual, intravenous, intramuscular or subcutaneous administration.
The pharmaceutical composition of the invention can be preferably directed to immediate, controlled or delayed release, more preferably for immediate release.
Moreover, the pharmaceutical composition of the invention is preferably administered once daily, twice daily, thrice daily, or according to the standard dose regime foreseen for the drug(s) contained in the composition .
The pharmaceutical composition of the invention shows the advantage of preventing and/or reducing the occurrence of idiosyncratic adverse reactions caused by those drugs containing at least one toxicophore function, by the addition of N-acetyl-L-cysteine .
As reported in the experimental part below, this advantage of the composition of the invention is demonstrated by the improved mitochondrial functionality
leading to an improvement of the hepatic cell growth ( hepatocytes ) .
In the evaluation of the hepatotoxicity of drugs containing a toxicophore function and giving rise to reactive quinone-imines , human hepatocytes were exposed to concentrations of diclofenac and/or nimesulide 60 to 240 times lower than those normally used with the control acetaminophen.
In these experiments mitochondria (MTS assay) were proved to be the primary hepatotoxicity target in living cells (LDH values unaffected) before ATP depletion occurs and without apparent evidence of oxidative stress (ROS values). Surprisingly the toxic effect of both drugs was reversed by almost equimolar amounts of N-acetyl-L- cysteine .
The invention also shows the advantage to provide a pharmaceutical formulation containing N-acetyl-L- cysteine with an improved stability.
According to a preferred embodiment of the invention, the pharmaceutical composition is formulated as an immediate release wherein the bioavailability rate of N- acetyl-L-cysteine is faster than that of the drug/s, contained in the pharmaceutical composition. These feature allows the N-acetyl-L-cysteine to enter the liver cells and the mitochondria before the drug thus maximizing the desired counteracting effect.
According to further preferred embodiments of the present invention, the composition comprises at least one of the following combinations:
Table 1 :
The composition according to the present invention and as above described preferably corresponds to a so-called fixed dose combination (FDC), as defined by "Guideline on clinical development of fixed combination medicinal products (CHMP) " London, 19 February 2009 doc. Ref . Chmp/ewp/240/95 rev.
According to the invention, and as indicated by EMEA, the term "fixed combinations" refers to medicinal products containing two or more active substances, which can be either well-known or not yet authorized for the intended claim, in fixed ratio of doses (see also WHO technical report series, N.929 p-no 107, 2005).
EXPERIMENTAL PART
Diclofenamic acid sodium salt (Diclofenac Na) ) ) , nimesulide, 4-Hydroxy-nimesulide and N- acetyl-L- cysteine were evaluated to determine the in vitro toxicity on human fresh hepatocytes.
The endpoints performed were: [ 3- ( 4 , 5-dimethylthiazol-2- yl ) -5- ( 3-carboxymethoxyphenyl ) -2- ( 4-sulfophenyl ) -2H- tetrazolium, (MTS) to determine mitochondrial functionality, adenosine triphosphate (ATP) levels to determine cellular energy levels, lactate dehydrogenase (LDH) release to determine membrane damage and reactive oxygen species (ROS) determination for reactive oxygen species .
The inhibitory activity of the compounds was calculated from dose-response curves and expressed as concentration inhibiting 50% cell growth (IC50) in treated cultures relative to untreated controls. For ROS detection the results were expressed as the percentage of increase or decrease of ROS values in treated samples compared to CTR values.
The IC50 values obtained for MTS endpoint were respectively 248.2 μΜ for nimesulide, 664.8 μΜ for 4-
Hydroxy nimesulide, 736.7 μΜ for diclofenamic acid sodium salt (Diclofenac Na) and >1000 μΜ for NAG
The IC50 values obtained for the ATP endpoint were of
198.4 μΜ for nimesulide, 496.4 μΜ for 4-Hydroxy nimesulide, 1108 μΜ for diclofenamic acid sodium salt
(Diclofenac Na) and >1000 μΜ for NAC, respectively.
No toxicity was observed regarding the LDH release, since all the compounds tested induced a percentage of extracellular LDH lower than 50% (i.e. the lower
threshold used to calculate the IC50) at the maximum concentration tested (1000 μΜ) .
For ROS detection, an increase of the reactive oxygen species compared to the specific controls (DMSO, PBS or distilled water) was observed for nimesulide starting from 250 μΜ, for diclofenamic acid sodium salt (Diclofenac Na) starting from 500 μΜ, while for 4- Hydroxy nimesulide only at the highest dose tested. For NAC the ROS detection demonstrated a reduction of the reactive oxygen species, when compared to the control, at all the doses tested.
Table 2: In Vitro Hepatotoxicity effect of Diclofenamic acid sodium salt (Diclofenac Na), Nimesulide , 4-Hydroxy Nimesulide and NAC in Primary Culture of Human Fresh Hepatocytes
MTS LDH
Compound ATP
IC50 μ M a)
IC50 uM a> ic50 μ M
a)
Diclofenamic
acid sodium >1000
salt 1108.0 736.7 (total LDH ( Diclofenac reduction )
Na)
>1000
Nimesulide 198.4 248.2 (total LDH
reduction )
4-Hydroxy >1000 nimesulide 496.4 664.8 (total LDH
reduction )
>1000
NAC >1000 >1000
>60
Acetaminophen
(mM) >60 26.13 (total LDH
reduction )
a) IC5o (concentration inhibiting 50% cell growth in treated cultures relative to untreated controls). 24 h treatment .
The results of the screening with nimesulide, 4-Hydroxy nimesulide, diclofenamic acid sodium salt ( Diclofenac Na ) and NAC on primary culture of human fresh hepatocytes showed a potential toxic effect of diclofenamic acid sodium( Diclofenac Na) , and a higher hepatotoxicity of nimesulide compared to its metabolite 4-Hydroxy nimesulide. No toxicity was observed for NAC for all the endpoints evaluated.
With the aim of assessing a protective effect of NAC on nimesulide, its metabolite and diclofenamic acid sodium salt ( Diclofenac Na) hepatotoxicity, a 2nd experiment was carried out taking ATP, MTS and ROS as endpoints.
The values obtained for the ATP were respectively of 46.24%, 55.61% and 77.75% of cell growth for nimesulide at the doses of 350, 250 and 150 μΜ in presence of NAC 800 μΜ ( Ihr of pre-incubation) . The data obtained in absence of NAC were 51.21%, 63.82% and 80.62% of cell growth at the same doses. These results demonstrated a dose related toxic effect of nimesulide, while the NAC addition did not prevent or reduce this toxicity.
Similar results were obtained for 4-Hydroxy nimesulide and diclofenamic acid sodium salt ( Diclofenac Na).
No toxicity was observed for the MTS endpoint in the 2nd experiment with nimesulide and 4-Hydroxy nimesulide administered alone, while a protective effect of NAC was seen on the toxicity induced by diclofenamic acid sodium salt ( Diclofenac Na) after a treatment of 750 μΜ on the human hepatocytes (37.95% of cell growth with
diclofenamic acid sodium salt ( Diclofenac Na) alone compared to 62.99% in presence of NAC).
In a 3rd experiment performed on MTS endpoint a marked toxic effect of nimesulide was observed on the human hepatocytes at all the tested doses in absence of NAC, with no dose proportionality (11.17%, 11.67 and 12.67% of cell growth). The addition of NAC (800μΜ) both with a pre-incubation of 1 hr and with the two test items added contemporaneously added, induced a protection from the toxic effect induced by nimesulide with a cell growth ranging from 27.84% to 29.98 (see Table 3).
Table 3: Effect of NAC on Nimesulide and Diclofenamic acid sodium salt ( Diclofenac Na) Hepatotoxicity in Primary Culture of Human Hepatocytes (24 h treatment)
a) % cell growth in treated cultures relative to untreated controls
b) 2 experiment
For ROS detection, a slight increase of reactive oxygen species compared to the specific controls (DMSO, PBS or distilled water) was observed for nimesulide at all the tested doses in presence and in absence of NAC at both the treatment times of 12 and 24 hours. For diclofenamic acid sodium salt (Diclofenac Na) a slight increase in ROS production was noted only after 24h of treatment, while only a minimal increase in ROS was observed with 4-hydroxy nimesulide at both treatment times. Overall, the presence of NAC did not show capability to protect against the test items toxicity, because a reduction of ROS detection was not observed when NAC was administered together with the tested compounds.
The positive controls, acetaminophen and pyocyanin, induced a marked dose-depended increase of ROS levels. The results of the screening with nimesulide and diclofenamic acid sodium salt (Diclofenac Na) in combination with NAC on primary culture of fresh human hepatocytes showed a protective effect of NAC on the hepatotoxicity induced by the test items with respect to the endpoint expressing the mitochondrial functionality, but not with ATP and ROS detection. No toxicity was observed for NAC when administered alone.
The following examples are included for better describing the invention, without limiting the same.
EXAMPLES
Example 1 : Nimesulide/NAC 100/200 immediate release conventional tablets
Example 2 : Nimesulide 100 /NAC 200 Sachets
Ingredients Function Amount % on
(mg) total weight
Nimesulide Active 100 5.00
NAC Active 200 10.00
Vitamin E Anti-oxidant 5 0.25
Lactose Diluent 60 3.00 monohydrate
Citric acid Aromatizer 5 0.25 anhydrous
Mannitol Diluent 1580 79.00
Na edetate Chelating agent 20 1.00
Sodium lauryl Disintegrant 10 0.50 sulphate
Orange powder Aromatizer 40 2.00
200 100
Example 3 : Diclofenamic acid sodium ( Diclofenac Na ) /NAC 50/100 immediate release bilayered tablets, 400 (Compression procedure)
Layer 1
Layer 2
Ingredients Function Amount % on
(mg) total weight
NAC Active 100 45.45
Vitamin E Anti-oxidant 5 2.27
Crosscaramellose Disintegrant 7 3.18
Silica colloidal Glidant 2 0.91 anhydrous
Cellulose Diluent 105 47.73 microcrystalline
Mg stearate Release 1 0.45 agent
220 100
Example 4 : Nimesulide/NAC 100/180 immediate release bilayered tablets, 510 mg (wet granulation procedure)
Layer 1
Layer 2
Ingredients Function Amount % on
(mg) total
weight
NAC Active 180 62.07
Vitamin E Anti-oxidant 5 1.72
Crosspovidone Disintegrant 4 1.38
Silica colloidal Glidant 1 0.34 anhydrous
Cellulose Diluent 92 31.72 microcrystalline
Mg stearate Release agent 2 0.69
PovidoneK30 Disintegrant 6 2.07
Example 5 : Diclofenamic acid sodium ( Diclofenac Na ) /NAC 75/150 vials (3 mL) for parenteral injection.
Differently from NAC, both nimesulide and diclofenamic acid sodium salt ( Diclofenac Na) are sparingly soluble in the gastric juice (pH 1-2) wheares they dissolve and become absorbable in the duodenum (pH>6). In the above mentioned formulations, this physico-chemical property delays their systemic availability with respect to NAC.
Example 6 : Dissolution test
Curve of mean dissolution values (% dissolved) of Nimesulide tablets (single layer) at different pH values .
Acetate Phosphate Phosphate
0.1 HC1
Time buffer buffer buffer
(min )
pH = 1
pH = 4.5 pH =6.8 pH = 8.2
10 7.04 8.07 22.24 74.72
20 7.82 8.67 25.71 85.34
30 8.00 8.96 27.37 89.59
45 8.28 9.15 28.79 92.43
60 8.46 9.21 29.69 93.40