BACKGROUND OF THE INVENTION
U is common knowledge that some active ingredients are having stability problems in solution. Some of these problems are due to the fact that the active ingredients easily oxidize, by reacting either with atmospheric oxygen or with dissolved oxygen in the aqueous solution, with consequent production of non-desirable degradation products.
Isoproterenol Hydrochloride is 3,4-Dihydroxy-a-[(isopropylamino)methyl] benzyl alcohol hydrochloride, a synthetic sympathomimetic amine that is structurally related to epinephrine but acts almost exclusively on beta receptors. Isoproterenol hydrochloride is a racemic compound.
Isoproterenol Hydrochloride Injection 0.2 mg/mL administered by intravenous infusion or bolus intravenous injection use in the treatment of bradycardia (slow heart rate), heart block, and rarely for asthma. Isoproterenol is a potent nonselective beta-adrenergic agonist with very low affinity for alphaadrenergic receptors. Intravenous infusion of Isoproterenol in man lowers peripheral vascular resistance, primarily in skeletal muscle but also in renal and mesenteric vascular beds.
Innovator product is available in the brand name of ISUPREL™ (Isoproterenol Hydrochloride) sterile Injection, USP in US market.
The sterile solution is nonpyrogenic and can be administered by the intravenous, intramuscular, subcutaneous, or intracardiac routes. ISUPREL M is supplied in 1 mL & 5ml ampoule 0.02% sterile, non-pyrogenic aqueous solution.
Catecholamines such as Isoproterenol and epinephrine are highly subject to degradative oxidation in solutions. During Drug product development stage, it is observed that, Isoproterenol HC injection in aqueous solutions is highly unstable when packed without control on dissolved

antioxidants fail to display the beneficial properties of stabilizing Isoproterenol. Among other antioxidants which have been found undesirable are butylatedhydroxy anisole. propyl gal late. nor-dihydroguaretic acid, sodium metabisulfite, ethyl hydrocatfeate. di-teributyl-paracresol and others.
The present inventors have carried out various researches in order to develop a Isoproterenol pharmaceutical formulation in the form of a solution for injection, having an improved stability.
The present inventors surprisingly found that, when headspace oxygen in the vials & ampoules filled with solution for injection was substantially removed (i.e., approximately less than 2%) by e.g.. bubbling of nitrogen gas into the solution & purging nitrogen gas over head space of vials/ ampoules after solution filling, a stable liquid formulation containing Isoproterenol or its sail is obtained.
Therefore, the present invention provides a method for preparing a pharmaceutical formulation in the form of a stable solution for injection containing Isoproterenol or its salt as an active ingredient.
The present invention overcomes the limitations of Isoproterenol solutions by stabilizing solutions of Isoproterenol by controlling the level of dissolved oxygen content and head space oxygen in the container.

In one aspect, the invention relates to a composition comprising a therapeutically effective amount of Isoproterenol in an aqueous solution having less than or equal to 2.0% oxygen in the headspace of a container when determined immediately after container sealing., said solution consisting essentially of a complexing agent and buffers. The compositions provide a stable low-oxygen formulation having less than or equal to 5 ppm of oxygen.
Yet another aspect of the invention relates to a process for preparing a low-oxygen composition of aqueous Isoproterenol, comprising the steps of:
Step-1: Add and dissolve batch quantity of complexing agent in 80% of water for injection at 60-65°C.
Step-2: To the step-1 solution add buffers and stabilizing agents at room temperature. Step-3: Nitrogen purge the bulk solution continuously to get dissolved oxygen below 5ppm. Step-4: Add and dissolve drug substance to step-3 solution and adjust the pH of bulk solution lo ■ 3.5 - 4.5 either by use of hydrochloric acid or sodium hydroxide. Step-5: Make up the volume to 100% WV with use of water for injection.
Step-6: Continuously nitrogen purge the bulk solutions to maintain dissolved oxygen below 5 ppm.
Step-7: Filter the bulk solution using 0.2 micron PVDF sterile filter membrane. Step-8: Fill the filtered solution in to 5 mL clear glass vial (USP type I glass) stoppered with coated rubber stopper and flip off seal. And ensure that the head space was blanketed with nitrogen gas.
The composition can be prepared in a vial according to steps (I) through (8) as described above. Appropriate means of closure was placed into the opening of the filled container in a manner which does not allow the exchange of gas from an interior side of the container to an exterior

DETAILED DESCRIPTION OF THE INVENTION
Drug stability means the ability of the pharmaceutical dosage form to maintain the physical chemical, therapeutic and microbial properties during the time of storage and usage by the patient. It is measured by the rate of changes that take place in the pharmaceutical dosage forms.
Stability is defined by the criteria set by ICH Ql A for injections.
The invention relates to the use of a stable Pharmaceutical composition of Isoproterenol.
It also relates to said composition characterized in that it is as a liquid stable Isoproterenol formulation.
Preferably the solution is an aqueous solution. The formulations as developed by the Inventors of the present Application are suitable for parenteral administration. Such stable liquid formulation of Isoproterenol can be developed with the use of tonicity agent, chelating agents and buffers and by controlling the oxygen content of drug solution and vial/ampoule headspace with the use of an inert gas viz nitrogen. These formulations are presented as a single unit dose (vial/ ampoule) presentation having Isoproterenol concentrations 0.2 mg/ml. These pharmaceutical compositions are then administered usually via intravenous infusion or bolus intravenous injection. In dire emergencies, the drug may be administered by intracardiac injection. If time is not of the utmost importance, initial therapy by intramuscular or subcutaneous injection is preferred.
The stable ready-to-use pharmaceutical composition of Isoproterenol is usually solvated in an aqueous solvent comprising water for injection. In one embodiment of the present invention, the ready-to-use pharmaceutical composition of Isoproterenol has a pH between about 2.5 and about

Deoxygenation and inerting is carried out by use of inert gas and preferably dense as nitrogen is circulated and / or sparging facilities and other tubing and solutions in order to eliminate most of any trace of oxygen and residual volumes in solution.
According to the invention the pharmaceutical composition is characterized by its oxygen content which is less than 5ppm in bulk solution and less than 5% in head space. In other embodiments and depending on the intended stability times, the oxygen content may be less than 5ppm in bulk solution and less than 2% in head space.
The gas used for purging the sealable container may be any appropriate inert gas known to those in the art. the most commonly used gases being argon, helium or nitrogen, or mixtures thereof. However the most preferred inert gas is nitrogen.
In another embodiment of the present invention, so as to minimize oxidation of the sensitive material it is also desirable to remove headspace oxygen from the sealable vessel as quickly as possible. This may be aided by, for example, purging the sealable container with a gas which is substantially oxygen-free, or substantially oxygen and moisture free before, during or after step. or any combination thereof. Purging can be expected to reduce the oxygen level in the scalable container to a level of from about 0%to about 5% typically about 2%or lower, depending on the efficiency of flushing and how quickly the container is sealed after flushing.
The invention is further illustrated by way of the following example, which in no way should be construed as limiting the scope of the invention.

Step-4: Add and dissolve drug substance to step-3 solution and adjust the pH of bulk solution to
3.5 - 4.5 either by useof dilute Hydrochloric acid or sodium hydroxide solution.
Step-5: Make up the volume to 100% V/V with use of water for injection.
Step-6: Continuously nitrogen purge the bulk solutions to maintain dissolved oxygen below
5ppm.
Step-7;Filter the bulk solution using 0.2 micron PVDF sterile filter membrane.
Step-8: Fill the filtered solution in to 5 mL clear glass vial (USP type I glass) stoppered with
coaled rubber stopper and flip off seal. And ensure that the head space was blanketed with

Note: Dissolved oxygen level less than 2 ppm and head space of ampoules were purged with nitrogen gas (preandpost N2 purging) as per the respective plan.
Table#4: Head space oxygen content measured by FMS- Oxygen Head space analyzer from
Lighthouse
Batch No. Batch Details Head space oxygen content (%)
IDRFD010-FD/042-026 Without head space 20.042
1DRFDO10-FD/042-027 2 sec head space 0.957
(DRFD010-FD/042-028 4 sec head space 0.335
IDRFD010-FD/042-029 6 sec head space 0.655
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Figure 1: Graphical representation of% impurity level Vs head space nitrogen time interval
Inference: From above stress condition (60°C) stability results, without head space nitrogen purged batch (head space oxygen content (20.042%) has more impurity formation compared to all other batches, Head space oxygen content is significantly affecting product stability. Hence final drug product should have low head space oxygen content (<2%) to maintain drug product stability throughout shelf life
Experiments performed to demonstrate the product instability when packed without head space nitrogen purging in 5mL glass vials. Experiments showed that without nitrogen head space samples packed in 5mL vials had higher total impurities, indicating thai head space oxygen plays major role in product stability, thus complete head space blanketing with nitrogen gas should be done to avoid degradation and to maintain product stability throughout shelf life.
The foregoing specification, including the specific embodiments and example, is proposed to be illustrative of the present invention and is not to be taken as limiting. Numerous other

1. A stable Pharmaceutical composition of Isoproterenol having head space oxygen content less than or equal to 5.0% and dissolved oxygen in solution is less than or equal to 5ppm.
2. A stable Pharmaceutical composition of Isoproterenol according to Claim 1 having head space oxygen content less than or equal to 2.0% and dissolved oxygen in solution is less than or equal to 5ppm.
3. A stable Pharmaceutical composition of Isoproterenol according to Claim 1 is liquid composition.
4. A stable Pharmaceutical composition of Isoproterenol according to Claim 1 wherein liquid composition is in an aqueous solvent.
5. A stable Pharmaceutical composition of Isoproterenol according to Claim I wherein the Isoproterenol formulation in said solvent is deoxygenated by flushing an inert gas into I he bulk solution during manufacturing and purging inert gas into the headspace of the final container.
6. A stable Isoproterenol aqueous formulation comprising excipients selected from stabilizer, complexing agent, buffering agent, tonicity agent, a base and an acid.
7. A stable Isoproterenol aqueous formulation according to claim 6 wherein the stabilizer is chelating agent and antioxidant.
8. A stable Isoproterenol aqueous formulation according to claim 6 wherein the chetalting agent is selected from the group consisting of ethylenediaminetetraacelic acid, diethylenetriaminepentaacetic acid, l,4,7,IO-tetraazacyc!ododecane-N,N',N",N'"-tetraacetic acid, trans-l,2-cyclohexylenediamine-N,N,N',N'-tetraacetic acid, N6 -carboxymethyl-N3\N9 - 2!3-dihydroxy-N-methy!propy!carbamoylmethyl!-3,6,9-triazaundecanedioic acid. N6 -

!6. A process for producing a liquid stable Isoproterenol aqueous formulation according to claims 14 wherein the inert gas is nitrogen.