DEVELOPMENT OF A TECHNOLOGY FOR PRODUCING A STABLE INJECTABLE DOSAGE FORM OF A HYDROPHOBIC INDOLOCARBAZOLE DERIVATIVE

Objective: Development of a technology for the production of a stable injectable dosage form (IDF) of indolocarbazole derivative LHS-1269. Methods: LHS-1269 is an active pharmaceutical ingredient that was synthesized in the N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation. The IDF includes dimethyl sulfoxide (DMSO), 95% ethanol, Kollidon® 17PF and water for injection. Magnetic stirrer and overhead stirrer with a propeller stirring element were used to prepare the model solution of the IDF of LHS-1269. Sterilizing filtration of the solution was performed with 0.2 −0.22 um polycarbonate, cellulose, polyvinylidene fluoride, poly ethersulfone and nylon membrane filters. The aqueous solution of LHS-1269 was lyophilized in Edwards Minifast DO.2 freeze dryer. Assay of LHS-1269 was performed by spectrophotometry at 320±3 nm. Potentiometry was used to measure pH, a viscosimetry method was used to measure the viscosity of the solutions. The average weight was estimated by weighing a sample of 10 vials with the concentrate. Results: 0.5% aqueous solution of LHS-1269 was produced by mixing the solution of the active substance in DMSO and ethanol with an aqueous solution of polyvinylpyrrolidone gradually at the ratio of LHS-1269/DMSO/ethanol/Kollidon® of 1/11/32/40 by weight. The aqueous solution of the study substance cannot be lyophilized, so a sequence of technological operations was presented to produce an anhydrous concentrate “LHS- 1269, concentrate for solution for injection and infusion 25 mg”. Conclusion: A technology was developed to produce a stable IDF of a hydrophobic indolocarbazole derivative LHS-1269, a high-potential antitumor drug.

Clinical studies of various indolocarbazole derivatives demonstrated a wide range of potential therapeutic indications and good compatibility with the known drugs. Indolo[2, 3-а]carbazoles have the most pronounced antitumor effect, in line with the antibacterial, antiparasitic and other types of biological activity. The antitumor effect is based on various mechanisms of action, including intercalation in DNA, inhibition of topoisomerases I and II, protein kinases C and A, cyclin-dependent kinases [2, 3].
The chemical synthesis laboratory of N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation developed an original technology to produce indolo[2, 3а]pyrollo [3,4-с]carbazole-5,7-dione N-glycosides ( fig. 1) with a cytotoxic and antitumor activity [4]. Up to date, this method has been used to synthesize and study more than 300 indolocarbazole derivatives. A few active compounds with different carbohydrate residues, including LHS-1269, were selected as antitumor drug candidates for the next phases of development.

L-arabinoses, D-riboses, D-xyloses) and hexoses (D-galactoses)
and R is amino group, benzamido group, (pyrid-2-yl)amino group, acetamido group, or urea group [3,4-с]carbazole-6-yl}pyridin-2-carboxamide, an original indolocarbazole derivative with xylose residue. This compound has a pronounced cytotoxic activity (IC50 1.7⋅10 -6 М) in the human colon cancer cell line НСТ-116, which is the most susceptible to drugs of this class. The studies in vivo showed a high and long-term antitumor effect on several mice models of continuous solid tumors: Lewis epidermoid lung carcinoma, AKATOL adenocarcinoma of the colon, CC-5 cervical cancer, Ca-755 mammary adenocarcinoma, B-16 melanoma, P-388 ascites lymphocytic leukemia, and Ehrlich ascites carcinoma [5,6]. Also, LHS-1269 was shown to have an antiangiogenic effect because it can inhibit vasculogenic mimicry of the tumor, i.e., formation of vascular canals by aggressive tumor cells with high-grade cell phenotype, such as melanoma, without endothelial cells and fibroblasts [7,8].
LHS-1269 substance is a yellow amorphous powder that is freely soluble in dimethyl sulfoxide, slightly soluble in acetone and I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f A Ap pp pl li ie ed d P Ph ha ar rm ma ac ce eu ut ti ic cs s benzene, very slightly soluble in ethanol and practically insoluble in water. Low solubility in water presents a challenge for developing the suitable dosage form, leads to inconsistent absorption and low systemic bioavailability. Parenteral administration (intravenous, intramuscular, subcutaneous and other) ensures a considerably high absorption profile and increased bioavailability. However, the low solubility of the drug substance makes intravenous administration of the solutions impossible and dangerous because it might lead to precipitation and vascular obstruction. Various physical, chemical and technological methods are used alone or in combination to increase the solubility and produce an injectable dosage form of a hydrophobic substance [9−11].
Objective of this study was to develop a technology to produce a stable injectable dosage form of LHS-1269.

Production of an injectable dosage form of LHS-1269
The solubilization, dissolution, filtration and freeze-drying steps were performed after preparing model solutions of the studied indolocarbazole derivative.

Quantitative analysis of LHS-1269 in the model solutions
Assay of the active substance was performed using spectrophotometry with a working reference standard (RS) at 320±3 nm.

Analysis of pH of LHS-1269 solution
The pH value of the samples of LHS-1269 dosage form was determined at 21 to 25 °С. Procedure: Add 8 ml of purified water to the concentrate and mix to obtain a homogenous solution and measure the pH value.

Analysis of viscosity of LHS-1269 solution
The dynamic viscosity of the samples of LHS-1269 dosage form was determined at 21 to 25 °С. Procedure: Add 2.9 ml of purified water to the concentrate and mix to obtain a homogenous solution with a 5 mg/ml concentration of the active substance. Mix the obtained solutions from 2 vials and measure the viscosity.

Estimation of an average weight of the vial contents
Ten filled vials with concentrate were sampled. The labels were removed, the vials were weighed individually. The contents were quantitatively removed from each vial, the vials were thoroughly rinsed inside. Each vial was dried and weighed again. The weight of the contents was calculated as the difference between the weight of the filled vial and the empty vial.

RESULTS AND DISCUSSION
While developing a water-soluble LHS-1269 dosage form, we considered a few pharmaceutical technological approaches that comply with the physical and chemical properties of the active substance. The approaches included solubilization/complexformation, production of solid dispersion, change of the pH/saltformation and inclusion into biocompatible phospholipid vesicles, i.e.,  Note. The data are given as mean±SD, n=3 The data analysis showed that the content of LHS-1269 in the solution is almost independent of the type of filter. The loss of the active substance was minimal with the use of nylon and polyethersulfone filters.
The solution of LHS-1269 becomes opaque and forms a precipitate after 3 d of storage at+4 °С. Since the model dosage form has low stability, the potential improvement of the stability by freeze drying was considered. Freeze drying of the solution of LHS-1269 was performed by long-term incubation at-45.-47 °С because the solution contains organic solvents with low eutectic points [12]. The sterile aqueous solution of LHS-1269 was dispensed in the portions of 3 ml into 20 ml vials, placed at the shelves of the freeze dryer and frozen at-47 °С. Then the drug product was incubated for around 12 h at this temperature. The vacuum pump was turned on afterward. In 3.5 h after equilibration of the vacuum, the temperature of the shelves was raised to-45 °С, the drug product was incubated at this temperature for 3.5 h. Them the shelf temperature was raised to-43 °С. At this point, a dome-shaped film appeared at the surface of the frozen mass. When the temperature of the drug product reached-35 °С, the mass began to break down. As a result, lyophilization was discontinued.
The high content of ethanol probably caused the difficulties with lyophilization of the studied dosage form of LHS-1269. According to the literature [13], alcohol affects the freeze drying of solutions significantly. Duration of the drying is prolonged if the solutions contain 10% of ethanol. The solutions with 20% ethanol develop an abnormal shape/structure during freeze-drying, the process is elongated considerably as a result. Various combinations of excipients of different origins (Kollidon® 17PF, Macrogol 400, and Poloxamer 188) were used as solubilizers to decrease ethanol concentration in the studied composition. This decrease, together with the increase of pH to slightly alkaline value, lowered solubility of LHS-1269 or therapeutic efficacy of the drug.
Freeze drying of the aqueous solution of LHS-1269 is not possible, so we propose producing a stable injectable dosage form of LHS-1269 as a concentrated mixture of drug substance, DMSO, 95% ethanol, and Kollidon® 17PF. The concentrate is dissolved in water for injection immediately before administration. Production of the dosage form LHS-1269, concentrate for solution for injection and infusion 25 mg, includes the following technological stages:   Note. Viscosity of the 5 mg/ml solution of LHS-1269 prepared by dissolving the concentrate in water for injection. The data are given as mean±SD, n=3.

CONCLUSION
The study led to development of a technology to produce a stable injectable dosage form of an original hydrophobic derivative of indolocarbazole LHS-1269. The aqueous solution of the studied indolocarbazole derivative has low stability and cannot be lyophilized, so an anhydrous concentrate of LHS-1269 was produced that is dissolved in water for injection immediately before administration. LHS-1269 is a high-potential antitumor drug. Our study showed that it could be formulated in a stable injectable dosage form convenient for clinical practice.