Field of the invention

The present invention relates to pharmaceutical formulations, in particular relate to a drug product of urate oxidase with the diluent.

Background of the Invention

Urate Oxidase (Uricase, EC (Enzyme commission) 1.7.3.4), an enzyme found in liver peroxisomes of most mammalian species , converts uric acid to a more soluble and easily excreted compound, i.e. Allantoin. Higher primates lack functional uricase and excrete uric acid as the end product of purine degradation. During chemotherapy to treat leukemia, lymphoma or other solid tumors, a marked increase in the excretion of uric acid levels derived from the nucleic acids of malignant cells can obstruct renal tubules, which leads to the formation of crystals of excess uric acid causing acute renal failure. This accumulation of uric acid or hyperuricaemia is part of constellation of other symptoms, medically termed as the tumor lysis syndrome constitutes a medical emergency necessitating immediate treatment of the symptoms. Part of that treatment includes attempts to reduce the uric acid.

Hyperuricaemia often proves difficult to control in some patients. Recombinant and non-recombinant Urate Oxidase is one treatment approved for the treatment of this particularly difficult clinical condition both in United States and Europe, amongst other Countries.
Urate Oxidase is a tetramer enzyme composed of four identical units. Each monomer unit formed from a single polypeptide chain containing 301 amino acids with a molecular weight of 34,152 Daltons and this subunit is acetylated at N-terminal site. The enzyme Urate Oxidase does not have disulfide bonds and its optimum pH for stability is 8.0. The cDNA for this protein has been cloned and expressed in E. coli, A.flavus and in S.cerevisiae.

The Urate Oxidase enzyme is readily oxidizable, which leads to the formation of higher aggregates and appears as a slightly turbid solution after reconstitution with diluents like water, buffer or Poloxamer. The formation of this turbidity and aggregation of protein in solution is accelerated by time, temperature and intensity of shaking the solution.

The aggregation of proteins is known to increase their immunogenicity. Unintentional aggregation of proteins has also been recognized as contributing factor for immunogenicity
during clinical use of therapeutic proteins. In order to obtain a clear solution of Urate Oxidase drug product after reconstitution, which can be administered to patients, there is constant search for a better diluent. The key to such diluents is that they must retain clarity, activity and biochemical integrity of the drug product under the suitable conditions of storage at the time of delivery.

GRAS (Generally Regarded as Safe) is a classification that lists substances that are generally regarded as safe by experts for the intended purpose of use. Such GRAS substances can be used for a long period of time without any toxic side effects. The diluent available for reconstitution of Urate Oxidase drug product in the market such as poloxamer is not GRAS listed. High intake of this may hazardous. Furthermore, poloxamer is not stable for longer periods as it tends to oxidize and hence an antioxidant additive, such as BHT or BHA are required to be added in the solution so as to increase the shelf life of the product. This is not suggestible as further additives need to be added to the reconstituent diluent (poloxamer).

Disadvantages in the use of poloxamer as a diluent component for reconstitution of proteins is listed below:

• It is not listed as GRAS.
• It is not stable for longer time (and toxic in the long run).
• Antioxidants are required for long term storage.
• Aqueous solutions of poloxamer may support mold growth.
• Not metabolized in the body and hence WHO recommends very less consumption.

Hence there is a need to reduce the quantity of poloxamer used as the diluents or substitute it with a suitable diluents that is safe (GRAS) and which would be stable for longer periods of time without addition of further additives or excipients. Accordingly, present invention aims at a composition with a diluent which is GRAS, stable without addition of further additives a such as antioxidants and one being easily metabolized in the body.

Object of the Invention

The main object of the present invention is to provide a lyophilized urate oxidase drug product with a suitable diluent for reconstitution of which retains clarity of the solution for extended periods of usage.

The other object of the invention is to provide a diluent such that after reconstitution, the solution maintains the desired activity of Urate Oxidase drug product.

Summary of the invention

The present invention provides a suitable diluent for solubilizing lyophilized Urate Oxidase drug product. The drug product comprises of urate oxidase and excipients such as alanine, mannitol and disodium hydrogen phosphate.

The diluent is a combination of GRAS listed Propylene Glycol and low amount of Polaxamer.
After reconstitution of lyophilized drug product with the diluent, the reconstituted solution retains clarity, without any aggregates, turbidity, viscosity and precipitation at least for 0-4 h at 4°CorO-4hat25°C.

After reconstitution of lyophilized drug product with the diluent, the reconstituted solution retains its Enzyme activity at least for 0-4 h at 4° C or 0-4 h at 25°C.

Detailed Description of the Invention

Accordingly, the present invention provides a Urate Oxidase drug product with a suitable diluent for reconstitution which aggregate free, and physically stable solution after reconstitution.

The diluent is a combination of GRAS listed Propylene Glycol and low amount of Polaxamer. After reconstitution of lyophilized powder of Urate Oxidase with the diluent provides an aggregate free, stable and active solution for at least 0-4h at 4°C or at 0-4h at 25°C.

The Urate Oxidase of the composition is produced in E.coli by recombinant process which is known to a skilled person in the art. The concentration of the drug product is in the range of 1 to 8 mg/mL.

Propylene glycol or a combination of propylene glycol and poloxamer in the ratio of 9:1 is used as the diluent in the composition, preferably the ratio of propylene glycol and poloxamer is 3:1, and most preferably 1:1 . Poloxamer (0.1% w/v) was used as a control.

The drug product comprises of urate oxidase and excipients such as 15.9 mg alanine, 10.6 mg mannitol and 12.6 - 14.3 mg disodium hydrogen phosphate.

The invention diluent used here is the combination of propylene glycol and poloxamer in the ratio of 3:1. On reconstitution of the urate oxidase drug product with the diluent of the present invention, the solution does not develop any turbidity or aggregation or precipitation at least 0-4h at 25°C or 0-4h at 4°C and also maintained the enzyme activity.

With 1:1 combination of propylene glycol and poloxamer, initially it appeared clear and colour less at both 4°C and 25°C. After one hour of reconstitution at 4°C & 25°C, no fibers appeared but slightly turbidity of solution equal to poloxamer was observed. After 2nd hour at 4°C & 25°C and turbidity was very high.

The diluent used with urate oxidase is a mixture of 0.1% Propylene Glycol (v/v) and 0.1% Poloxamer (w/v).

Advantages of the composition of the present invention

1. The diluent of the present invention is mixture of GRAS listed propylene glycol and poloxamer suitable for administration for a longer period of time, without any toxic side effects.
2. The diluent is stable and hence there is no need for the addition of further excipients/additives such as antioxidants in the composition.

3. The diluent is a good preservative and also prevents the growth of microbes for a longer period of time in comparison with the diluents that are commercially available for Urate Oxidase.

4. After reconstitution of Urate Oxidase with the diluent,

a. The solution is clear, with no turbidity.

b. The solution is stable for without any aggregation or precipitation or turbidity for a period of 0- 4 h at both 4°C and 25°C.

c. The biological activity of the reconstituted Urate Oxidase drug product is maintained during its stable period.

Examples

The present invention is described with respect to the following examples, which are provided merely for illustration and are not intended to restrict the scope of invention in any manner.
Any embodiments that may be apparent to a person skilled in the art are deemed to fall within the scope of present invention.

Example 1:

Step 1: Preparation of the diluent and reconstitution of the Lyophilized drug product using the diluent:

Stock preparation:

0.1% Propylene Glycol (v/v):

Dissolved 0.1ml of propylene glycol in 90ml of water for injection and made up to 100 ml.
0.1% Poloxamer (w/v):

Dissolved O.lg of Poloxamer in 90ml of water for injection and made up to 100 ml.

Preparation of 9:1 diluent:

A mixture of 0.1% Propylene Glycol (v/v) and 0.1% Poloxamer (w/v) was prepared by mixing in the ratio 9:1. One ml of prepared diluent was added to the lyophilized vial containing 1.5 mg of Urate Oxidase and excipients such as 15.9 mg alanine, 10.6 mg mannitol and 12.6 - 14.3 mg disodium hydrogen phosphate,gently mixed until the powder got dissolved.

Preparation of 1:1 diluent:

A mixture of 0.1% Propylene Glycol (v/v) and 0.1% Poloxamer (w/v) was prepared by mixing in the ratio 1:1. One ml of prepared diluent was added to the lyophilized vial containing 1.5 mg of Urate Oxidase and gently mixed until the powder got dissolved.

Preparation of 3:1 diluent:

A mixture of 0.1% Propylene Glycol (v/v) and 0.1% Poloxamer (w/v) was prepared by mixing in the ratio 3:1. One ml of prepared diluent was added to the lyophilized vial containing 1.5 mg of Urate Oxidase and gently mixed until the powder got dissolved.

Step 2: Physical appearance:

The reconstituted vials of step 1, which were stored at 25°C and at 4°C were examined for physical appearance (colour and particles) under the white and black background. The data is provided in Table 1 & 2.

Step 3: Measurement of turbidity:

The reconstituted vials of step 1, which were stored at 25°C and at 4°C were tested for turbidity by measuring the absorbance/OD at 360nm using spectrophotometer. The data is provided in Table 1 & 2.

Step 4: Measurement of Urate Oxidase activity:

The reconstituted vials of step 1, which were stored at 25°C and at 4°C were examined for the enzymatic activity at different time intervals after reconstitution. The enzymatic activity of the Urate Oxidase drug product is measured by using the specific substrate i,e uric acid. Uric acid in presence of enzyme will be converted to Allontoin. Native uric acid generally absorbs UV light at 293 nm. The reduction of the substrate concentration in presence of Urate Oxidase enzyme is determined spectrophotometrically at 25°C by monitoring the disappearance of uric acid (substrate) at 293nm (Legoux et al., J. Biol. Chem. 1992, 267(12) 8565-8570). Data is provided in Table 1&2.

The reconstituted vials of the present invention maintain the enzymatic activity for the said period.

Example 2:

Preclinical toxicity studies were conducted using Urate Oxidase drug product (source of urate oxidase -recombinant produced by Virchow Biotech Private Limited) with the diluent to evaluate the safety of the recombinant drug product.

Acute Toxicity Studies: Single dose acute toxicity studies were conducted by administering 10 X (High dose (HD)) doses of recombinant urate Oxidase drug product with the diluent on Swiss Albino Mice (18.2 mg/kg body weight) and Wistar Rats (12.6 mg/kg body weight). The first group of 20 Mice (10 Males+10 Females) received the reconstituted drug which was administered through subcutaneous and intravenous routes and the animals were observed for the period of 14 days for mortality and morbidity. The second group of 20 Rats (10 Males+10 Females) received the drug through intravenous and subcutaneous route of administration and observed the animals for a period of 14 days for mortality and morbidity if any. No pre terminal mortality or morbidity was observed through both routes of administration in this study.

Sub chronic toxicity studies: Repeat dose sub chronic toxicity studies were conducted on 48 Swiss Albino Mice and 24 New Zealand White Rabbits. All the animals were divided into 4 groups and different dose levels (Therapeutic Dose 0.65 mg/kg Body Weight for Rabbits and 1.82mg/kg for Mice, Average Dose 3.2 mg/kg BW for Rabbits and 9.1mg/kg for Mice, High Dose 6.5 mg/kg BW for Rabbits and 18.2 mg/kg for Mice) of recombinant Urate Oxidase was administered for 5 consecutive days through intravenous route of administration. One group of animals were kept as control and treated with vehicle. The animals were observed for a period of 28 days after the last administration of the drug. Parameters evaluated including clinical signs of toxicity, in life phase of animals, cage side observations and body weight and food consumption. Blood samples were collected on days 14 and 28 for hematology and clinical chemistry analysis. Animals were necropsied and vital organs were weighed and subjected to histopathological examination. Bone marrow samples were collected and evaluated for Geno & Cytotoxicity.

The results of these studies clearly indicate that, there was no pre terminal morbidity or mortality was observed after repeated administration of the test compound. All clinical chemistry and hematological parameters were found to be normal and within the physiological range. Antibody response for the administration of the test compound was nil and no neutralizing antibodies were found in the serum of High Dose (HD) group animals. Over all
Recombinant Urate Oxidase

(source-Virchow Biotech) with the diluent was well tolerated and no toxicological impact of the drug was observed even after 10X (18.2 mg/kg for mice and 6.5 mg/kg for rabbits) dose administration.

Sub chronic Toxicity study design:

Example 3: Clinical summary of Urate Oxidase drug product with the diluent

In a clinical trial the Urate Oxidase drug product (source of urate oxidase -recombinant produced by Virchow Biotech Private Limited) with the diluent was evaluated for its safety and efficacy on Indian patients with high risk for TUmour Lysis Syndrome (TULS). The clinical trial was an open-label multicentre study conducted at 4 centers. A total of 80 patients with high risk for tumour lysis syndrome were screened, and 56 eligible patients participated in the study. During the study period of 10 days, 8 patients were lost to follow-up.

Among the 56 patients, who participated in the study, there were 19 children (age <18 years) and 37 adults (>18 years). Majority of patients (58.9%) had ALL and the remaining had other hematologic malignancies such as AML (21.4%), NHL (8.9%), CLL (7.2%) and APL (3.6%). Majority of patients well tolerated administration of urate oxidase and as a result, there was an excellent compliance to treatment and 85.7% of participating patients completed the study.
Safety: There were no serious adverse events except in two patients during or after administration of TULY. The study of various safety parameters like vitals, haematology and biochemical parameters showed that there were no significant changes in any of the parameters of clinical significance throughout the study period of 10 days except an improvement in reduction of plasma uric acid levels and LDH levels.

Safety of TULY was assessed on the basis of changes in vitals, hematological and biochemical test results from baseline to termination. Safety results clearly indicated that there were no significant changes in any of the vitals, hematological and biochemical test results from baseline to termination, except in plasma uric acid and LDH levels. Changes in plasma uric acid levels are expected as uric acid lowering therapy drug TULY was administered daily for 4 days. Similarly, since LDH levels reflect tumor mass, changes in its levels can be explained on the basis of tumor size reduction in response to chemotherapy. Though 22 adverse events, which included fever, headache, rigors, were reported in 12 patients, none of the patients required hemodialysis due to renal failure, which is a common complication of TLS.
Efficacy: The outcome measures were percentage of reduction in plasma uric acid at 4 hrs after intravenous administration of TULY, plasma uric acid AUCo-% hr and incidence of adverse events. In 73.3% patients plasma uric acid levels returned to normal level by 4 hrs after TULY therapy and in the remaining by 12-36 hrs. After TULY administration, there was 74.9 ± 26.5% reduction in plasma uric acid at 4 hrs over baseline. The plasma uric acid AUC 0-96 hr was 255 ± 174 mg/dl.hr.

The open-label, multi-centre clinical trial demonstrated that the present urate oxidase is safe and effective for prevention and management of hyperuricemia in both children and adults who are at high risk of developing TLS.

Advantage of the Invention:

The oral toxicity of propylene glycol is very low, and large quantities are required to cause perceptible health damage in humans. Serious toxicity will occur only at plasma concentrations over 4g/L, which requires extremely high intake over a relatively short period of time. The use of propylene glycol as diluent for reconstitution of lyophilized protein further increase the stability of protein by preventing aggregation and it is advantageous in formulations due to its non toxic activity and stabilizing activity.

In the pre-existing reconstitution diluent present in the market (poloxamer) for urate oxidase drug product intake level of polaxamer is approximately 14mg/70kg body weight per day. The present invention diluent reduces the polaxamer quantity to 3.5mg/70kg body weight per day.
A low level of poloxamer intake is acceptable by WHO.


We Claim

1. A pharmaceutical composition of lyophilized urate oxidase drug product with a diluent wherein the diluent comprises of propylene glycol and poloxamer 188 in the ratio of 9:1.

2. The pharmaceutical composition as claimed in claim 1, wherein the ratio of propylene glycol and poloxamer 188 is preferably in the ratio of 3:1.

3. The pharmaceutical composition as claimed in claim 1, wherein urate oxidase drug product is in the range of 1.5 -8 mg/ml of diluent.

4. The pharmaceutical composition as claimed in claim 1, wherein the urate oxidase drug product comprises of urate oxidase and exepients such as alanine, mannitol and disodium hydrogen phosphate.