FORMULATION AND EVALUATION OF SOMATOSTATIN ANALOGUE TABLETS

Objective: This study was undertaken with the aim of the formulation and evaluation of hypothalamic hormone somatostatin analogue tablets, which are intended for neuroendocrine tumours treatment. 
Methods: Tablets were prepared by using the wet granulation method and evaluated for weight variation, resistance to crushing, friability, disintegration time, content and content uniformity. Stability tests have been performed. 
Results: Characteristics of somatostatin analogue cyphetrylin powder have been investigated, and the wet granulation method has been chosen to prepare tablets. Technical pharmaceutical properties of formulations of different compositions and obtained tablets were estimated and the best formulation CF11 has been established with appropriate characteristics: disintegration time less than 15 min, resistance to crushing more than 30 kg·m·s−2, weight variation ˂7.5 %, active substance content closed to the nominal quantity and consistent cyphetrylin distribution in the batch. Performed examinations showed cyphetrylin tablets stability in the long-term study. 
Conclusion: Because of somatostatin analogue cyphetrylin powder properties tablets were prepared by the wet granulation method with starch, lactose, povidone, talc, microcrystalline cellulose, calcium stearate and magnesium stearate as excipients. The best formulation including povidone has characteristics corresponding to requirements of European Pharmacopoeia (PhEur) and State Pharmacopoeia of the Russian Federation (PhRu). Examination of cyphetrylin tablets showed its quality and stability within the time of observation–12 mo.


INTRODUCTION
Neuroendocrine Tumours (NET) are rare human tumour arising from the cells of the diffuse neuroendocrine system located in various organs and tissues of the human body (gastrointestinal tract, lungs, pituitary gland, etc.). This type of tumours is characterized by the presence of secondary symptoms caused by metabolically active substances hyperproduction and secretion [1,2].
Biotherapy with Somatostatin Analogues (SSA) has a proven effect in the treatment of clinical syndromes (carcinoid syndrome, hypoglycemia syndrome, Zollinger-Ellison syndrome, etc.). The drugs in this group (most widely used three SSA are as follows: octreotide, lanreotide and pasireotide) suppress clinical symptoms and reduce the level of biochemical tumour markers and also have useful anti-proliferative activity [3,4].
Several SSAs has been synthesised in N.N. Blokhin NMRCO, including cytotoxic SSAs, and their antitumor effect has been studied in the experiments in vitro and in vivo. According to the results of the preliminary investigations, a hypothalamic hormone somatostatin analogue cyphetrylin has been selected for a preclinical study. Cyphetrylin is pentapeptide (Boc-Cys(Thp)-Phe-D-Trp-Lys(Z)-Thr-OMe) with noncyclic structure [5].
Usually, parenteral way of administration is used for peptide drug administration because of their enzymatic instability [6,7]. But cyphetrylin is insoluble in water, and it is metabolically stable in the gastric fluid because of reactive groups blocking. The specific studies of cyphetrylin in vivo showed its high antitumor efficiency on animal's experimental tumours and xenografts by oral administration [8,9].
The objective of the present study was to create hypothalamic hormone analogue cyphetrylin dosage form composition and preparation method and to evaluate obtained pharmaceutical by different parameters according to requirements of current editions of PhEur and PhRu [10,11].
This investigation is the first attempt to obtain a dosage form of new original somatostatin analogue for oral administration.

Materials
Cyphetrylin substance was obtained from chemical synthesis laboratory of N.N. Blokhin NMRCO, potato starch, lactose (because cyphetrylin isn't sensitive to moisture we used lactose monohydrate [12]), povidone, talc, microcrystalline cellulose, calcium stearate and magnesium stearate were purchased from different suppliers and conformed to PhEur requirements. All other used chemicals and solvents were of analytical grade.

Bulk density
2 g of cyphetrylin powder or cyphetrylin granulate previously lightly shaken to break any agglomerates formed was introduced into a 10 ml measuring cylinder. After the initial volume was observed, then calculate the bulk density using the following formula [10,11].
Bulk Density = Weight of the powder Bulk volume

Angle of repose
The angle of repose of cyphetrylin granulate was determined by the fixed funnel method. The accurately weighed powder was taken in a funnel. The height of the funnel was adjusted in such a way that the tip of the funnel just touched the apex of the heap of the powder. The powder was allowed to flow through the funnel freely onto the surface. The diameter of the powder cone was measured, and the angle of repose was calculated using the following equation [10,11].

Weight variation test
To study weight variation 20 tablets of each formulation were weighed using an electronic analytical balance. The average weight was calculated, then individual tablet weight was compared with average weight [10,11].

Disintegration time
The examination of disintegration time was carried on randomly selected six tablets of each formulation without disks. The purified water was used as a disintegration medium and temperature was maintained at 37±0.5 °C. The average disintegration time of six tablets was noted down for calculation [10,11].

Resistance to crushing and friability
The test of resistance to crushing was measured on tester consists of two jaws facing each other, one of which moves towards the other. The tester is calibrated with the precision of kg·m·s −2

An analytical method for cyphetrylin assay in tablets
. The measurement is carried out on ten tablets [10,11].
The friability test was performed by using tablet friability tester Erweka (Germany). Ten tablets were accurately weighed and placed in the tester and operated for 100 revolutions. The tablets were dedusted and reweighed, and friability was calculated using the following equation.

Friability =
Initial weight − Final weight Initial weight x 100 The analytical method for cyphetrylin assay in tablets has been created and validated [13,14]. Cyphetrylin content under formulation developed was estimated with Carry 100 UV-Visible spectrophotometer (USA). The specially synthesised cyphetrylin substance of 99.0 % of the active ingredient was used as a standard sample.
About 100 mg of cyphetrylin rubbed tablets were dissolved in 50 ml of ethanol (0.12 mg/ml) and filtered via Millipore with pore size 0.45 μm. The absorbance of this solution was measured at the maximum wavelength of cyphetrylin of 282±2 nm. The absorbance of the cyphetrylin standard spirituous solution of the same concentration was measured in parallel. Cyphetrylin quantity calculation was carried out by a formula taking into consideration dilutions of all solutions and cyphetrylin content in substance.

Uniformity of drug content
The determination was performed for ten tablets separately by UVspectrophotometry method described for assay test. 25 ml of 95 % ethyl alcohol were added to each tablet and shook intensively during 8-10 min. The liquid was filtered, and the drug content was determined after appropriate dilution with 95 % ethyl alcohol.

Characterization of powder
The general technological properties of cyphetrylin powder mentioned in table 1. It is visible that cyphetrylin powder is polydisperse-its particles are of different form and size (table 1). The fraction with a particle size less 0.25 mm is more than half, that way it is necessary to use excipients to increase particle size and mass and flowability. The substance doesn't get wet, and it means that it is required to include appropriate excipients to control tablets disintegration.
To improve cyphetrylin powder properties (at first flowability) we decided to use wet granulation. Because cyphetrylin is insoluble in water, but the mixture of cyphetrylin and excipients is well wettable by water, 5% starch paste and 1% and 2% povidone solution were tested as granulation agents.
To prepare granulate of cyphetrylin the mixtures of active substance and excipients (for 20 tablets) were wetted by starch paste or 1% or 2 % povidone solution and dried to complete drying out. When dried, granulates were passed through a mesh sieve #710, powder mixture (magnesium stearate, talc and starch) was added to the blend and compressed into tablets on eccentric tablet press Erweka (Germany) using 6 mm punch.
Formulations with the different correlation of ingredients have been prepared (table 2). The cyphetrylin dose in all tablets was 6 mg. As it was shown by a series of biological experiments in vivo, it is required cyphetrylin quantity to demonstrate antitumor efficiency and safety. The flow properties of the final blend were tested by measuring bulk density, flowability and the angle of repose and they were found to be the best for CF11 formulations. As is seen from table 3, povidone inclusion into formulation led not only to sufficient flow characteristics but to the optimal correlation between resistance to crushing and disintegration.
Experimental tablets were prepared by tablet press Erweka (Germany). All the prepared formulations were evaluated for different physicochemical properties for physical parameters: appearance, resistance to crushing, friability, disintegration, average weight, weight variation, and results are presented in table 4. Appearance and test of weight variation may be the most straightforward indicators of technology compliance. The appearance of prepared cyphetrylin tablets was high-quality (white, straight, circular, 6 mm diameter, biconvex tablets) except formulations CF2, CF3 and CF9, which surfaces were off-white and slightly marbled (of unequal colour).
The examination of weight variation showed that average weight of cyphetrylin tablets of all compositions was within the range of calculated mass±10 %, and maximum weight variation was 7.29%, which was in PhEur and PhRu limits of±7.5%.
Tablet mechanical characteristics provide their stability within the manufacture and useful quality. Tablets must pass these points, and tablet defects are nonaccepted.
The results of prepared tablets in the test of resistance to crushing were in the range of 7.32 to 38.32 kg·m·s −2 but only composition CF11 resistance to crushing (38.32±0.12 kg·m·s −2 ) corresponded to PhRu recommendations-minimal value has to be not less than 30 kg·m·s −2 The friability test for compositions CF2, CF3, CF6 and CF9 was more than the limit (1 %), but other compositions friability was less what is illustrative of tablets good mechanical resistance.
for tablets of this diameter. At the same time, disintegration test for CF11 composition was successful, and tablets passed the test (less than 15 min).
All formulations passed the disintegration test by taking time (not more than 15 min). Formulations CF4, CF6 and CF10 have shown disintegration time near to the limit, but formulations CF2 and CF9 had disintegration time lower than ideal. *All values are expressed as mean±SD, n=6, *n=20, #n=10 As is seen from table 4 above, investigations revealed that CF11 formulation with povidone addition has an optimal correlation of mechanical properties and disintegration.

Assay and content uniformity
Because cyphetrylin dose in the tablet is 6 mg (less than 6 %), the quantity of the active substance in each tablet is the most critical parameter. And according to the requirement of PhEur and PhRu, it is necessary to determine the uniformity of drug content. The test was carried out on ten tablets by direct estimation of each unit. The conditions of determination were equal to the assay test.
Results of quantitative determinations of two cyphetrylin tablets batches are presented in table 5. Each point is an average of six determinations. As it is seen from table 5, tests have shown the consistency of active substance content in the tablet and cyphetrylin distribution in the batch.