COMBINATION OF AN AMINOGLYCOSIDE ANTIBIOTIC AND A PARP
INHIBITOR FIELD OF THE INVENTION
The present invention is of a composition of matter, use thereof and treatment therewith for at least reducing or substantially alleviating, if not eliminating, toxicity associated with aminoglycoside antibiotics, and more preferably of a combination of an aminoglycoside antibiotic and a PARP inhibitor, use thereof and treatment therewith.
BACKGROUND OF THE INVENTION
Aminoglycoside antibiotics are extensively used as a therapy for gram negative bacterial infections. Although they have significant therapeutic advantages over other antibiotics, they also have potentially severe nephrotoxic effects (1). Gentamicin, the most common used aminoglycoside antibiotic, is responsible for acute renal failure in 10-20% of treated patients (2). Treatment for more than 7 days with gentamicin is associated with nephrotoxicity that leads to cessation of therapy in 30% of patients (3). Various changes to the pattern of administration of the drug (e.g. once per day instead of several times a day) did not result in substantial reduction of nephrotoxicity (4). The typical renal lesion caused by gentamicin is severe acute tubular necrosis (ATN) of the proximal tubule (5). Although the pathogenic mechanism is not absolutely clear, it seems that production of destructive reactive oxygen species (ROS) is intensified as a toxic effect of gentamicin, which may lead to nephrotoxicity (6).
Recent evidence supports the role of ROS in mediating the cellular life and death cycle in both in vivo and in vitro studies (7). ROS are responsible for processes which lead to cell death in various renal pathologic conditions such as glomerular diseases, renal ischemia with reperfusion injury and several types of toxic insults causing acute renal failure (8, 9, 10). Unfortunately, there is currently no known mechanism in the art to minimize the cellular damage in ATN and to encourage tissue regeneration (11).
In the ischemic tissue injury model, the damage to DNA is attributed to production of ROS, including superoxide anions, peroxynitrite, nitric oxide and hydrogen peroxide, which are produced in the ischemic tissue during the reperfusion phase. Without wishing to be limited by a single hypothesis, it is possible that gentamicin nephrotoxic activity causes a similar insult to DNA as a result of ROS production.
DNA injury features single or double strand breaks and, as a consequence, a chromatin bound enzyme, poly (ADP-ribose) polymerase (PARP), is activated and initiates the ribosylation process, binding ADP-ribose polymers to nuclear proteins and thus making the DNA repair enzyme accessible to the DNA-injured sites. PARP activation causes intracellular nicotinamide adenine dinucleotide waste to accumulate; returning the levels of such waste to normal amounts is an energy-consuming process for the cell. Thus, processes which cause significant DNA damage and hence lead to over activation of PARP can significantly reduce cellular energy levels, as a result of continuous efforts to cope with the consequences of accelerated poly (ADP- ribosylation) levels. This cascade of events can lead to cell death (12). PARP itself is a mediator of cell apoptosis.
Unfortunately, currently there are no known treatments and/or preventatives for aminoglycoside antibiotic induced nephrotoxicity.
SUMMARY OF THE INVENTION
The present invention overcomes the drawbacks of the background art by featuring a combination of an aminoglycoside antibiotic and a PARP inhibitor, as a composition of matter, a method of treatment and/or a use thereof.
By "combination" it is meant that both the (one or more) aminoglycoside antibiotic(s) and the (one or more) PARP inhibitor(s) are provided to the subject in need of treatment thereof, which is preferably a mammal in need of treatment thereof and more preferably a human in need of treatment thereof. Such provision may optionally be made simultaneously or substantially simultaneously, but may optionally be made sequentially or substantially sequentially and/or through a combination of simultaneous and sequential administration.
According to preferred embodiments of the present invention, the aminoglycoside antibiotic may optionally be administered in combination with any inhibitor of poly (ADP-ribose) polymerase activity, more preferably an inhibitor of PARP-I activity.
According to other preferred embodiments of the present invention, the aminoglycoside antibiotic(s) is preferably administered with the PARP inhibitor(s) simultaneously, optionally and preferably through a route of administration selected from one or more of paxenterally and/or topically. For example, if administered intravenously, optionally and preferably both the aminoglycoside antibiotic(s) and the PARP inhibitor(s) are provided in a single solution or other formulation which is suitable for intravenous administration. Alternatively or additionally, if administered intramuscularly, optionally and preferably both the aminoglycoside antibiotic(s) and the PARP inhibitor(s) are provided in a single formulation which is suitable for intramuscular administration, for example in a depot formulation as is known in the art. Also alternatively or additionally, if administered topically, optionally and preferably both the aminoglycoside antibiotic(s) and the PARP inhibitor(s) are provided in a single formulation which is suitable for topical administration, for example as a cream, patch, lotion, paste and/or other formulation as described in greater detail below.
If administered simultaneously, the combination may optionally be administered in a single formulation and/or a plurality of formulations. If administered sequentially, the combination may optionally be administered through a plurality of routes of administration.
"Parenteral" administration includes but is not limited to administration through any type of injection, or infusion, including but not limited to intravenous, intraarterial, intramuscular, subcutaneous, intraosseous infusion, intraperitoneal, epidural or intrathecal, and preferably includes intravenous and/or intramuscular administration.
"Topical" administration includes but is not limited to epicutaneous, inhalationaL rectal, optical, through the ear, intranasal, vaginal, transdermal or application to any mucosal membrane. An aminoglycoside antibiotic according to the present invention may optionally and preferably be selected from the group including, but not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin or a combination thereof. Neomycin and kanamycin are preferably administered orally or topically; the remaining aminoglycosides are preferably administered parenterally. The present invention is preferably operative with any aminoglycoside according to a "class effect" for this group of antibiotics.
Aminoglycoside antibiotics are also referred to herein as "aminoglycosides". Examples of bacterial infections which may optionally be treated according to the present invention include but are not limited to: aerobic gram-negative bacteria, including but not limited to bacilli and staphylococci. Non-limiting examples of such bacteria include but are not limited to Enterobacteriaceae, Pseudomonas SPP and Hemophilus influenzae. Additional or alternative examples of bacterial infections which may optionally be treated according to the present invention include but are not limited to: gram positive bacteria, including but not limited to methicillin-susceptible S aureus MSSA.
Without wishing to be limited in any way, a number of aminoglycoside antibiotics may optionally and preferably be used for particular types of infections. A number of illustrative, non-limiting examples are given herein. Gentamicin, tobramycin, amikacin, and netilmicin are preferably used against Pseudomonas aeruginosa. Gentamicin is preferably used against tuberculosis. Streptamycin is preferably used against brucellosis, tularemia, and plague. Gentamycin and tobramycin may optionally be used against Serratia SPP. Garamycin and tobramycin may optionally be used against P aeruginosa. Non-limiting examples of conditions or diseases for which the present invention may optionally and preferably be used include septicemia, nosocomial respiratory tract infection, complicated urinary tract infection and/or osteomyelitis.
Administration patterns and dosage levels for the above aminoglycosides could easily be determined by one of ordinary skill in the art. The combination may also optionally comprise a further antibiotic (other than an aminoglycoside) as could easily be determined by one of ordinary skill in the art.
For example, a beta-lactam antibiotic may optionally be combined with an aminoglycoside antibiotic according to the present invention for treatment of an infection selected from the group including but not limited to, Serratia spp, Pseudomonas spp, indole positive proteus, Citobacter spp, Acinetobacter spp or Enterobacter spp. Garamycin is preferably used in combination with such an antibiotic for invasive enterococcal infection and/or serious staphylococcal infection. Vancomycin or ampicillin (in combination with at least aminoglycoside) are preferably used for prophylactic treatment of ERCP or for those at risk of development of infective endocarditis.
A PARP inhibitor according to the present invention preferably comprises any suitable non-toxic PARP inhibitor, preferably a PARP 1 inhibitor, including but not limited to, 3AB (3-Aminobenzamide), KU-59436 (KuDOS Pharmaceuticals, United Kingdom), 4-Amino-l,8-naphthalimide, 4-HQN, 1,5-Isoquinolinediol, BGP -15, as well any PARP inhibitor which has shown to be active for other treatment applications, such as against cancer for example.
The combination of the present invention preferably at least reduces or alleviates, and more preferably eliminates, nephrotoxicity, which may optionally and more preferably comprise any type of impaired kidney function and/or kidney damage, optionally as measured by one or more of the following parameters: serum urea blood level, creatinine blood level, urinary protein excretion levels, urine TIA (trypsin inhibitory activity) levels; the number of tubuli with inner diameter greater than 20 microns; the number of infiltration foci; the number of granular cast- containing-tubuli; the number of cells containing enlarged nucleoli (macro nucleoli, above 5 micron); the number of PCNA-positive-staining nuclei; and the number of mitoses.
BRIEF DESCRIPTION QF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:
Figures 1 and 2 show serum urea and creatinine blood levels;
Figure 3 shows urinary protein excretion levels;
Figure 4 shows urine TIA (trypsin inhibitory activity) levels;
Figure 5 shows the amount of tubuli with inner diameter greater than 20 microns;
Figure 6 shows the number of infiltration foci;
Figure 7 shows the number of granular cast-containing-tubuli;
Figure 8 shows the number of cells containing enlarged nucleoli (macro nucleoli, above 5 micron); Figure 9 shows the number of PCNA-positive-staining nuclei; and Figure 10 shows the number of mitoses.
BRIEF DESCRIPTION OF THE TABLES The invention is also herein described, by way of example only, with reference to the accompanying tables. With specific reference now to the tables in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention.
In the Tables:
Tables Ia and Ib related to protein levels in the urine;
Table 2 provides information regarding GLM repeated measures between subject effects;
Table 3 relates to urine TIA (trypsin inhibitory activity); and
Table 4 shows that a statistical significance was found for the differences in both PCNA and macro nucleoli numbers.
DETAILED DESCRIPTION OF THE INVENTION
Aminoglycoside antibiotic agents, such as gentamicin for example, are extensively administered as a therapy for gram negative infections. This treatment is associated with nephrotoxicity in 10-20% of the patients, causing cessation of treatment in 30% of them. Without wishing to be limited by a single hypothesis, it is possible that this is a result of reactive oxygen species (ROS) production, which causes DNA destruction and thus, activation of poly(ADP-ribose) polymerase (PARP). Again without wishing to be limited by a single hypothesis, it is possible that through this process, a decline in nicotinamide adenine dinucleotide (NAD) causes an attenuation of the cellular energy capacity, leading to tubular cell death by necrosis. Acute tubular necrosis is therefore the end result of this cascade.
Again without wishing to be limited by a single hypothesis, it is possible that aminoglycoside-induced ATN (acute tubular necrosis), which causes oxidative stress ROS production and DNA injury, similar to the insult in ischemic model, may be at least reduced, if not substantially alleviated or even eliminated through the administration of PARP inhibitors. Again without wishing to be limited by a single hypothesis, such inhibitors may attenuate tubular cellular damage and thereby accelerate the regeneration and healing process.
Experiments were performed as described in greater detail below, in which the administration of an aminoglycoside antibiotic, gentamicin, and a PARP inhibitor, 3 AB, together resulted in significant protection against nephrotoxicity as compared to the effects of gentamicin alone. Again without wishing to be limited by a single hypothesis, these results support the important role of PARP in proliferation, apoptosis and necrosis processes.
EXAMPLE l
The effect of PARP inhibitor on gerxtamicin-induced nephrotoxicity in rats was studied as a non-limiting, illustrative example of nephrotoxicity induced by administration of an aminoglycoside antibiotic. Twenty female Wistar-Kyoto rats were divided to 4 groups: group 1 was the control; group 2 was treated with a PARP- inhibitor alone (10 mg/kg of 3 -amino berizarnine (3 AB) as an exemplary, non- limiting, illustrative PARP inhibitor); group 3 was treated with gentamicin alone as an exemplary, non-limiting, illustrative aminoglycoside antibiotic, at a dose of 80 mg/kg); and group 4 was treated with a combination of the PARP inhibitor 3 AB and gentamicin, at the same doses of each substance as groups 2 and 3, respectively. During the twelve days of the study, blood and urine were examined for parameters relating to kidney function, protein level and gentamicin level. In addition, urine was tested also for trypsin inhibitory activity (TIA). Urinary trypsin inhibitory activity is a measure of kidney damage; it rises as damage increases through to end stage renal failure (Inhanced Urinary Trypsin in Gentamycin Induced
Nephrotoxicity in Rats, S. Smetana, Clinica Chimica Acta 176(1988) 333-342).
At the end of the study, the rats were sacrificed and their kidneys were examined for overall pathology, as well as for immunohistochemistry related to proliferating cell nuclear antigen (PCNA). PCNA is a 36 kDa molecular weight protein also known as cyclin. The protein has also been identified as the polymerase- associated protein and is synthesized in early Gl and S phases of the cell cycle. In early S phase, PCNA has a very granular distribution and is absent from the nucleoli. At late S phase, PCNA is prominent in the nucleoli. In cells fixed with organic solvents, PCNA is seen to be strongly associated in the nuclear regions where DNA IL2007/001293
synthesis is occurring. For this study, PCNA was used as a marker for cell proliferation.
Results showed that impaired kidney function was found in group 3 (gentamicin alone), expressed by increased serum creatinine level in the fourth day (1.01+0.8 mg/dl) with no significant recovery after 12 days (0.95+0.22 mg/dl), compared to groups 1 and 2 (control and PARP inhibitor alone, respectively), in which serum creatinine levels were 0.62 - 0.63 mg/dl throughout the study. Group 4 (gentamicin plus PARP inhibitor) demonstrated a slight increase in serum creatinine concentration during the first 8 days, but this level returned to normal by the end of the study (0.63+0.6 mg/dl and 0.60+mg/dl, respectively).
Pathology and immunohistochemical examinations revealed diffuse tubular necrosis and significant regenerative changes in the kidneys of rats of group 3 but only relatively minor signs of tubular necrosis and mild regenerative changes in group 4. Kidneys of groups 1 and 2 were normal. Urine gentamicin excretion levels over 24 hours were high in groups 3 and 4 (495 to 26000 meg) and almost undetectable in groups 1 and 2 as expected, since only groups 3 and 4 received gentamicin.
These results show that administration of the PARP inhibitor 3 AB with gentamicin as a combined treatment significantly attenuates gentamicin-induced nephrotoxicity in the rat. Materials and methods, and results, are described in greater detail below.
Materials and methods
Twenty Wistar-Kyoto female rats, weighing 190-25Og, were kept hi metabolic cages and were divided to 4 groups, after an adaptation period of 7 days as follows: group 1 was the control; group 2 was treated with a PARP-inhibitor alone (10 mg/kg of 3-amino benzamine (3 AB) as an exemplary, non-limiting, illustrative PARP inhibitor); group 3 was treated with gentamicin alone as an exemplary, non-limiting, illustrative aminoglycoside antibiotic, at a dose of 80 mg/kg); and group 4 was treated with a combination of the PARP inhibitor 3 AB and gentamicin, at the same doses of each substance as groups 2 and 3, respectively.
The PARP inhibitor, 3 AB (Sigma-Aldrich), was dissolved in 1 ml NaCl 0.9% for each dose and was administered by intraperitoneal injection. Gentamicin sulphate from Biogal Pharmaceutical Works (Teva, Israel), was administered by intraperitoneal injection. Both gentamicin and 3 AB were administered on all of the days of the study.
The study was performed for 12 days. Blood samples were drawn on days 1, 4, 8 and 12. Urine was collected and measured on a daily basis. Blood samples were examined for urea and creatinine concentrations. Urine was examined for levels of excreted protein, trypsin inhibitory activity (TIA) and gentamicin level.
Blood samples were drawn by means of direct intracardiac puncture. Rats were anesthetized by ether during all invasive procedures. Blood samples were drawn without anticoagulant on the first day of the adaptation period and on the 4th, 8th and 12th days of the study in microtainer serum separator tubes. After centrifugation at 4 C, serum was separated and stored at -700C until analyzed. Continuous urine was sampled every day and stored at -700C until analyzed. At the end of the study, rats were sacrificed and kidneys were examined for pathology and immunohistochemistry for proliferative cell nuclear antigen (PCNA).
Laboratory methods: Concentrations of creatinine and urea in plasma and urinary excretion of creatinine and protein were measured with an Olympus AU2700 analyzer, using the manufacturer's kits.
Gentamicin levels in blood and urine were measured by fluorescence polarization immunoassay (FPIA), from AxSYM Abbott Laboratories Diagnostics, USA.
Trypsin inhibitory activity (TIA) in urine was assayed as described by Smethana et al (5). The rate of hydrolysis of p-tosyl-L-arginine-methyl-ester (TAME) (Sigma T-4626) was measured by the increase in absorbance at 247nm during 1 minute at 250C on a Spectronic 601 spectrophotometer (Milton Roy Company,
Rochester, N. Y. USA). Inhibition units were expressed as microliter urine needed to achieve 50% inhibition of 4 microgram trypsin. Trypsin inhibitory activity in the urine was expressed as inhibition units per day.
Histology and immunohistochemistry: histology and immunohistochemistry were performed using 5-μm sections of 4% formalin-fixed paraffin-embedded kidneys. Four kidney sections from each rat stained with hematoxylin and eosin were assessed to determine the morphological changes. The sections were viewed under 10 power lens and 25 randomly selected images from subcapsular-cortical non- overlapping fields were captured on a computer. The following parameters were evaluated: (a) the number of tubules with an inner diameter exceeding 20 μm per 1 square-millimeter field, (b) the number of mitoses per 1 square-millimeter field, (c) the number of foci within the interstitium composed of more than ten mononuclear inflammatory cells per 1 square-millimeter field, (d) the number of tubules containing granular casts per 1 square-millimeter field and (e) the number of cells with macronucleoli (larger than 5 μm in diameter) per 1 square-millimeter field.
Immunohistochemistry for Proliferating Cell Nuclear Antigen (PCNA) was performed using PCNA specific antibody (DAKO) and basic aminoethyl carbazole (AEC) detection kit with Ventana automatic stainer (see for example Localization Of Proliferating Cell Nuclear Antigen; Vimentin; C-Fas; and Clustering in Post- Ischemic Kidney, Witzgall R. et aL J. Clin Invest. 93 ;2175-2188; 1994). Four kidney sections from each rat stained with PCNA immunostain were viewed and captured on a computer as mentioned above and the number of unequivocally positively stained nuclei per 1 square-millimeter field was determined.
Data analysis: analysis of data was carried out using SPSS 9.0 statistical analysis software (SPSS Inc., Chicago, IL, USA, 1999). For continuous variables, such as chemistry parameters, descriptive statistics were calculated and are reported as mean + standard deviation. Normalcy of distribution of continuous variables was assessed using the Koknogorov-Smirnov test (cut off at p=0.01). The t-test for independent samples was used to compare continuous variables by treatment condition. All tests were two-sided and considered significant at p<0.05.
Power calculation and sample size: with a sample size of 12 rats per group, the present study was designed to have 80% power to detect a true, by treatment difference in serum urea of 49+40 mg/dl using the t-test for independent samples, assuming a two-sided alpha of 0.05. This sample size also provides 80% power to detect true by-treatment assignment difference in serum creatinine of 0.3+0.2 mg/dl.
Serum and urine parameters measured repeatedly over time were compared using general linear modeling (GLM) repeated measures analysis, into which treatment group was entered as a fixed factor.
Results
Serum urea and creatinine blood levels are shown in Figures 1 and 2. Rats of group 3 (gentamicin -treated) had elevated levels compared to those of groups l(control) and 2 (3AB-treated) during the entire study. In group 4 (gentamicin + 3AB- treated) the results were very similar to samples from rats having normal levels of urea and creatinine, without significant differences as compared to the results from groups 1 and 2. Urine protein excretion was significantly elevated in group 3, starting from the second day until the end of the study. Moderate proteinuria was seen in group 4, with a trend of decline in the last study days. Groups 1 and 2 had normal urinary protein excretion (Figure 3). The increased proteinuria seen in group 3 (gentamicin alone) compared to group 4 (gentamicin plus 3 AB) was statistically significant on days 3, 6 and 11 (Tables 1 a and Ib), using also GLM repeated measures between subject effects (Table 2).
Urine gentamicin levels are extremely high in groups 3 and 4 (>40 mcg/24 hours), compared to groups 1 and 2 (<0.3 mcg/24 hours), as expected since only groups 3 and 4 received gentamicin. Urine TIA is significantly higher in group 3 compared to group 4, in which a slight elevation is seen (Figure 4). This difference is statistically significant (Table 3). Histopathology and immunohistochemistry results (all parameters are related to an area of 10 mm2) are as follows. The amount of tubuli with inner diameter greater than 20 micron is shown in Figure 5. In both groups 3 and 4, the results are significantly elevated above normal (as determined according to results from the control group, group 1). In group 3 the amount is almost twice that of group 4.
Figure 6 shows the number of infiltration foci: group 4 had the same number of foci as group 2, five, which is only slightly above the control group (0). hi group 3 there were 26.25 foci, a significant increase. Figure 7 shows the number of granular cast-containing-tubuli, which was high in both group 3 and 4, with a 5-fold higher result for group 3. In groups 1 and 2, such tubuli are not found at all.
Figure 8 shows the number of cells containing enlarged nucleoli (macro nucleoli, above 5 micron): Null (zero) in group 2, 18.3 in control group, moderate increase in group 4 and extremely high in group 3 (43.3 and 197.5, respectively).
Figure 9 shows the number of PCNA-positive-staining nuclei: in group 3 more than 16-fold and in group 4 less than 3 -fold of the level of groups 1 and 2. Figure 10 shows the number of mitoses. No mitoses were found in group 2, 12 in group 1, whereas 3585 and 626 mitoses were found in groups 3 and 4, respectively.
Table 4 shows that a statistical significance was found for the differences in both PCNA and macro nucleoli numbers between group 3 and group 4.
Discussion
Extensive necrotic damage was found in the kidney tissue of group 3, based on the finding in histology slides and also on the multitude of PCNA-positive nuclei, which is a well accepted criterion for high proliferation and regeneration rates, which are proportional to the amount of necrosis as these are symptoms of previous tissue damage (5). Additional supportive findings are the increased counts of macro nucleoli, dilated tubules, granulated casts and mitoses in kidneys of group 3, but not in control and 3 AB-treated groups. All the mentioned above changes are present to a much lower extent in group 4 (gentamicin+3AB). The differences in PCNA-positive nuclei and cellular macro nucleoli counts between groups 3 and 4 are statistically significant.
Gentamicin levels are well above the maximal levels which are considered to be toxic in groups 3 and 4 and non-detectable in groups 1 and 2. The kidney function deterioration together with increased urine protein excretion and TIA levels with significant histopathology and immunohistochemical changes in group 3, but not in groups 1 and 2 and only moderate changes in group 4, are consistent with protective effect of the PARP inhibitor 3 AB on gentamicin nephrotoxicity in the studied rats. The lack of statistical significance for certain of the criteria may stem from the limited size of the study groups.
The results show a clear-cut effect of the PARP inhibitor on the biochemical and histological consequences of gentamicin nephrotoxicity, by pronounced attenuation of tubular damage. Acceleration of regeneration and proliferation processes by this agent causes a complete recovery of kidney functions at the end of the study (12 days), following transient slight to moderate impairment.
Combining the administration of a PARP inhibitor with gentamicin administration significantly lessens gentamicin nephrotoxicity in the rat.
EXAMPLE 2 Preventive effect of PARP inhibition on aminoglycoside-induced acute tubular necrosis in rats
Gentamicin, an aminoglycoside-antibiotic used to treat gram negative infections, causes nephrotoxicity in 10-20% of patients by generating reactive oxygen species (ROS), leading to DNA destruction and activation of poly(ADP-ribose) polymerase (PARP). The ensuing decline in nicotinamide adenine dinucleotide (NAD) causes diminished cellular energetic capacity and necrotic tubular cell death. This Example seeks to prevent such damage through the administration of a PARP inhibitor as a preventive for kidney damage in a model of aminoglycoside- induced acute tubular necrosis (ATN) in rats.
Materials and Methods: Ten (n=10) female Wistar-Kyoto rats will be divided into treatment groups: control (no treatment or treatment with PARP inhibitor Pj34); gentamicin-sulphate (80 mg/kg); or gentamicm-sulphate + PJ34, doses as described for other treatment groups. The planned follow-up is 12 days, not including a seven-day adaptation period. Blood samples will be drawn on days 1, 4, 8 and 12. Urine will be collected and measured daily. Blood samples will be examined for urea and creatinine concentrations. Urine will be examined for protein excretion, tripsin inhibitor activity (TIA) and gentamicin level. Pj34 will be introduced by intraperitoneal injection on a daily basis once the treatment period starts. Gentamicin sulphate will be introduced by intraperitoneal injection on a daily basis once the treatment period starts as well. The treatment with Pj34 will be initiated concomitantly with gentamicin, and will be continued until the end. of follow-up on day 12. At the end of the study, rats will be sacrificed and their kidneys will be examined for pathology and immunuhistochemistry for proliferative cell nuclear antigen (PCNA).
EXAMPLE S Treatment of aminoglycoside-induced acute tubular necrosis present in rats with
PARP inhibitor As noted above, gentamicin, an aminoglycoside-antibiotic used to treat gram negative infections, causes nephrotoxicity in 10-20% of patients by generating reactive oxygen species (ROS), leading to DNA destruction and activation of poly(ADP-ribose) polymerase (PARP). The ensuing decline in nicotinamide adenine dinucleotide (NAD) causes diminished cellular energetic capacity and necrotic tubular cell death.
This Example seeks to treat such damage after it has already occurred, as well as to optionally prevent further damage, through the administration of a PARP inhibitor as a preventive for kidney damage in a model of aminoglycoside-induced acute tubular necrosis (ATN) in rats.
Materials and Methods: Ten (n=10) female Wistar-Kyoto rats will be divided into treatment groups: control (no treatment or treatment with PARP inhibitor Pj34); gentamicin-sulphate (80 mg/kg); or gentamicin-sulphate + PJ34, doses as described for other treatment groups. The planned follow-up is 12 days, not including a seven-day adaptation period before treatment begins. Blood samples will be drawn on days 1, 4, 8 and 12. Urine will be collected and measured daily. Blood samples will be examined for urea and creatinine concentrations. Urine will be examined for protein excretion, trypsin inhibitor activity (TIA) and gentamicin level. Pj34 will be introduced by daily intraperitoneal injection. Gentamicin sulphate will be introduced by daily intraperitoneal injection as well. The treatment with Pj34 will be initiated 72 hours after gentamicin treatment is begun, at which point kidney damage will be measurable, and will be continued until the end of follow-up on day 12. At the end of the study, rats will be sacrificed and their kidneys will be examined for pathology; immunuhistochemistry studies will be performed for proliferative cell nuclear antigen (PCNA).
EXAMPLE 4 TREATMENT WITH THE PRESENT INVENTION
The combination of an aminoglycoside antibiotic and a PARP-I inhibitor according to the present invention is suitable for treatment of a variety of bacterial infections.
The method of treatment (and/or use of the combination of the present invention) optionally and preferably comprises: providing the combination of aminoglycoside antibiotic and PARP inhibitor to a subject in need of treatment thereof. The substances may optionally be administered sequentially or substantially sequentially, simultaneously or substantially simultaneously, or with a combination thereof. The combination may optionally comprise a plurality of such antibiotics and/or a plurality of such PARP inhibitors. If administered simultaneously, the combination may optionally be administered in a single formulation and/or a plurality of formulations. If administered sequentially, the combination may optionally be administered through a plurality of routes of administration. An aminoglycoside antibiotic according to the present invention may optionally and preferably be selected from the group including, but not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin or a combination thereof. Neomycin and kanamycin are preferably administered orally or topically; the remaining aminoglycosides are preferably administered parenterally. The combination may also optionally comprise a further antibiotic (other than an aminoglycoside) as could easily be determined by one of ordinary skill in the art.
A PARP inhibitor according to the present invention preferably comprises any suitable non-toxic PARP inhibitor, preferably a PARP 1 inhibitor, including but not limited to, 3AB (3-Aminobenzamide), KU-59436 (KuDOS Pharmaceuticals, United Kingdom), 4-Amino-l,8-naphthalimide3 4-HQN, 1,5-Isoquinolinediol, BGP -15, PJ- 34 (N-(6-Oxo-5,6-dmydro-phenanthridin-2-yl)-N,N-dimethylacetamide . HCl), INO- 1001 (inotek Corp) as well any PARP inhibitor which has shown to be active for other treatment applications, such as against cancer for example.
By "mammalian subject" or "mammalian patient" is meant any mammal for which gene therapy is desired, including human, bovine, equine, canine, and feline subjects, most preferably, a human subject.
It should be noted that the term "treatment" also includes amelioration or alleviation of a pathological condition and/or one or more symptoms thereof, curing such a condition, or preventing the genesis of such a condition. Examples of bacterial infections which may optionally be treated according to the present invention include but are not limited to: aerobic gram-negative bacteria, including but not limited to bacilli and staphylococci. Non-limiting examples of such bacteria include but are not limited to Enterobacteriaceae, Pseudomonas SPP and Hemophilus influenzae. Additional or alternative examples of bacterial infections which may optionally be treated according to the present invention include but are not limited to: gram positive bacteria, including but not limited to methicillin-susceptible S aureus MSSA.
Without wishing to be limited in any way, a number of aminoglycoside antibiotics may optionally and preferably be used for particular types of infections. A number of illustrative, non-limiting examples are given herein. Gentamicin, tobramycin, amikacin, and netilmicin are preferably used against Pseudomonas aeruginosa. Gentamicin is preferably used against tuberculosis. Streptamycin is preferably used against brucellosis, tularemia, and plague. Gentamycin and tobramycin may optionally be used against Serratia SPP. Garamycin and tobramycin may optionally be used against P aeruginosa.
Non-limiting examples of conditions or diseases for which the present invention may optionally and preferably be used include septicemia, nosocomial respiratory tract infection, complicated urinary tract infection and/or osteomyelitis. Administration patterns and dosage levels for the above aminoglycosides could easily be determined by one of ordinary skill in the art.
The combination may also optionally comprise a further antibiotic (other than an aminoglycoside) as could easily be determined by one of ordinary skill in the art.
For example, a beta-lactam antibiotic may optionally be combined with an aminoglycoside antibiotic according to the present invention for treatment of an infection selected from the group including but not limited to, Serratia spp, Pseudomonas spp, indole positive proteus, Citobacter spp, Acinetobacter spp or Enterobacter spp. Garamycin is preferably used in combination with such an antibiotic for invasive enterococcal infection and/or serious staphylococcal infection. Vancomycin or ampicillin (in combination with at least aminoglycoside) are preferably used for prophylactic treatment of ERCP or for those at risk of development of infective endocarditis.
The combination of the present invention can optionally be used to produce a pharmaceutical composition. Thus, according to another aspect of the present invention there is provided a pharmaceutical composition which includes, as active ingredients thereof, an aminoglycoside and a PARP inhibitor in a pharmaceutically acceptable carrier. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein, with other chemical components such as traditional drugs, physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the combination to an organism. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. In a preferred embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Hereinafter, the phrases "physiologically suitable carrier" and "pharmaceutically acceptable carrier" are interchangeably used and refer to an approved carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered conjugate.
The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the protein, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should be suitable for the mode of administration.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate processes and administration of the active ingredients. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Further techniques for formulation and administration of active ingredients may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference as if fully set forth herein.
The pharmaceutical compositions herein described may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the active ingredients can be optionally formulated for administration of one or both ingredients according to the present invention. The active ingredients can also be formulated by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the active ingredients of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-celmlose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.
Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols, hi addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide, hi the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the active ingredient and a suitable powder base such as lactose or starch.
The active ingredients described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, pharmaceutical compositions for intravenous adrninistration are solutions in sterile isotonic aqueous buffer. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
The active ingredients of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
The pharmaceutical compositions herein described may also comprise suitable solid of gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols. The topical route is optionally performed, and is assisted by a topical carrier. The topical carrier is one which is generally suited for topical active ingredient administration and includes any such materials known in the art. The topical carrier is selected so as to provide the composition in the desired form, e.g., as a liquid or non- liquid carrier, lotion, cream, paste, gel, powder, ointment, solvent, liquid diluent, drops and the like, and may be comprised of a material of either naturally occurring or synthetic origin. It is essential, clearly, that the selected carrier does not adversely affect the active agent or other components of the topical formulation, and which is stable with respect to all components of the topical formulation. Examples of suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like. Preferred formulations herein are colorless, odorless ointments, liquids, lotions, creams and gels.
Ointments are semisolid preparations, which are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum active ingredients delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton,
Pa.: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water- soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight; again, reference may be made to Remington: The Science and Practice of Pharmacy for further information.
Lotions are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations, in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in- water type. Lotions are preferred formulations herein for treating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as active ingredients useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethylcellulose, or the like.
Creams containing the selected active ingredients are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation, as explained in
Remington, supra, is generally a nonionic, anionic, cationic or amphoteric surfactant.
Gel formulations are preferred for application to the scalp. As will be appreciated by those working in the field of topical active ingredients formulation, gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.
Various additives, known to those skilled in the art, may be included in the topical formulations of the invention. For example, solvents may be used to solubilize certain active ingredients substances. Other optional additives include skin permeation enhancers, opacifiers, anti-oxidants, gelling agents, thickening agents, stabilizers, and the like.
The topical compositions of the present invention may also be delivered to the skin using conventional dermal-type patches or articles, wherein the active ingredients composition is contained within a laminated structure, that serves as a drug delivery device to be affixed to the skin. In such a structure, the active ingredients composition is contained in a layer, or "reservoir", underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during active ingredients delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. The particular polymeric adhesive selected will depend on the particular active ingredients, vehicle, etc., i.e., the adhesive must be compatible with all components of the active ingredients- containing composition. Alternatively, the active ingredients-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing material should be selected so that it is substantially impermeable to the active ingredients and to any other components of the active ingredients-containing composition, thus preventing loss of any components through the upper surface of the device. The backing layer may be either occlusive or non-occlusive, depending on whether it is desired that the skin become hydrated during active ingredient delivery. The backing is preferably made of a sheet or film of a preferably flexible elastomeric material. Examples of polymers that are suitable for the backing layer include but are not limited to polyethylene, polypropylene, and polyesters.
During storage and prior to use, the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the active ingredients reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin. The release liner should be made from an active ingredients/vehicle impermeable material.
Such devices may be fabricated using conventional techniques, known in the art, for example by casting a fluid admixture of adhesive, active ingredients and vehicle onto the backing layer, followed by lamination of the release liner. Similarly, the adhesive mixture may be cast onto the release liner, followed by lamination of the backing layer. Alternatively, the active ingredients reservoir may be prepared in the absence of active ingredients or excipient, and then loaded by "soaking" in an active ingredients/vehicle mixture. As with the topical formulations of the invention, the active ingredients composition contained within the active ingredients reservoirs of these laminated system may contain a number of components. In some cases, the active ingredients may be delivered "neat," i.e., in the absence of additional liquid. In most cases, however, the active ingredients will be dissolved, dispersed or suspended in a suitable pharmaceutically acceptable vehicle, typically a solvent or gel. Other components, which may be present, include preservatives, stabilizers, surfactants, and the like.
It should be noted that the combination of the present invention is preferably administered to the patient (subject) in need in an effective amount. As used herein, "effective amount" means an amount necessary to achieve a selected result. For example, an effective amount of the composition of the invention may be selected for being useful for the treatment of abacterial infection or disease.
Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any active ingredient used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from activity assays in animals. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined by activity assays.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the IC50 and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject active ingredient. The data obtained from these activity assays and animal studies can be used in formulating a range of dosage for use in human. For example, therapeutically effective doses suitable for treatment of genetic disorders can be determined from the experiments with animal models of these diseases. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, termed the minimal effective concentration (MEC). The MEC will vary for each preparation, but may optionally be estimated from whole animal data.
Dosage intervals can also be determined using the MEC value. Preparations may optionally be administered using a regimen, which maintains plasma levels above the MEC for 10-90 % of the time, preferable between 30-90 % and most preferably 50-90 %.
Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition described hereinabove, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising an active ingredient of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. References:
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