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Analysis of Ge-132 and development of a simple oral anticancer formulation

Analysis of Ge-132 and development of a simple oral anticancer formulation Volume 4 † Number 2 † June 2011 10.1093/biohorizons/hzr015 Advance Access publication 6 May 2011 ......................................................................................................................................................................................................................................... Research article Analysis of Ge-132 and development of a simple oral anticancer formulation Sara M. Ogwapit* School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AH, UK. * Corresponding author: Email: s.o.malinga@gmail.com Supervisors: Dr Katja Strohfeldt-Venables and Dr Clare Rawlinson Malone, School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, UK. ........................................................................................................................................................................................................................................ The anticancer activity of synthetic organogermanium, b-or bis-carboxyethylgermanium sesquioxide (Ge-132), has been demonstrated in several cancer cell models and human studies. Ge-132 increases pro-inflammatory responses by enhancing interferon-g (IFN-g), natural killer cell and T-cell activity, and is significantly less toxic than other widely used metal-based anticancer drugs such as cisplatin. In this small-scale laboratory study, we effectively assessed the physicochemical characteristics and purity of Ge-132, our main objective being to develop a novel oral anticancer formulation, using conventional tabletting excipients which do not alter the chemistry of Ge-132. We determined that solid Ge-132 decomposes at 3308C; is virtually insoluble in most common organic solvents; and readily 1 13 dissolves in water (saturation solubility 1.28 g/100 ml) to form germane triol (pH 3.06–3.12). H and C nuclear magnetic resonance spectroscopy confirmed the structure of our compound showing two identical proton environments at 1.55 and 2.65 ppm (triplets) and three distinct carbon environments at 178.31, 27.37 and 12.93 ppm. The mass spectrum indicated the formation of numerous complex ion fragments with masses ranging from m/z 123.1 to m/z 478.3. FT-infrared and FT-Raman spectra showed characteristic sesquioxide 21 21 peaks at 900.51, 900.26 and 800.04 cm and, most importantly, confirmed the absence of toxic, inorganic GeO , at 850 cm . While parenteral formulations exist for many anticancer medicines, here we successfully developed uncoated tablets containing Ge-132 (5% w/w) by manual direct compression (powder particle size 180 mm). The tablets passed British Pharmacopoeia (BP) content uni- formity testing (Ultraviolet–visible, 212 nm), and BP disintegration testing in both acidic and basic media, disintegrating between 2 min 55 s and 3 min 10 s, respectively. We prepared gastro-resistant formulations using Eudragit ; however, these failed content uniformity tests and had lower disintegration times (1 min 36 s), indicating that compatibility of polymers with Ge-132 requires further investi- gation. The results presented here support further larger-scale research on Ge-132 as a novel metal-based oral anticancer drug which can be conveniently administered alone or included within a chemotherapy regimen. Future formulation studies on Ge-132 could focus on compatibility assessments with nano-formulations in keeping with current advancements in metal-based anticancer therapies. Key words: Ge-132, spectral analysis, anticancer, oral formulation. Submitted September 2010; accepted February 2011 ........................................................................................................................................................................................................................................ platinum (II) and platinum (IV) complexes which inhibit Introduction 5,6 cell division. Cisplatin is one of the most successful metal- Despite advancements in cancer research and drug delivery, based anticancer drugs effective against a diverse range of many cancers still do not have adequate targeted treatments. tumour types, though it is associated with significant renal Modern anticancer therapies show improved clinical efficacy toxicity and induced or acquired resistance. The search for and specificity towards cancer cells; however, their success is novel anticancer drugs continues and other metals have 7,9–20 limited by the development of resistance and numerous side been investigated for their anticancer potential, includ- 2–4 effects, complicating their use in chemotherapy regimens. ing germanium (Ge), a naturally occurring metalloid found Metal-based anticancer therapy arose from the discovery of in soil. ......................................................................................................................................................................................................................................... The Author 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited 128 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Ge is a constituent of many plants, including garlic and aquated and activated within the cell cytoplasm require ginseng root, and may play a role in photosynthetic, self- sufficient protection to bypass healthy cells and target defence and metabolic processes. The daily intake of cancer cells. Although there are several novel formulations 44–46 elemental Ge from food is estimated between 367 and of successful platinum-based drugs, there are limited 3700 mg and both organic and inorganic Ge compounds formulation studies of other metal-based anticancer drugs have been used for many years as a dietary supplement in (including organogermanium compounds) into viable drug doses of 15 mg–1 g per day. Various synthetic organic delivery forms as most organometallic chemistry does not Ge compounds have been investigated for their therapeutic incorporate formulation science. efficacy in osteoporosis, malaria, hypertension, cancer and Parenteral drug administration is limited to hospitals or 24–29 AIDS. One of the most widely researched is the sesqui- specialized treatment centres where patients rely on trained oxide, b-or bis-carboxyethylgermanium sesquioxide, also professionals using complex equipment. Additionally, par- known as Ge-132, an organogermanium synthesized from enteral formulations require sterile manufacturing conditions 30 48 the chloroform, HGeCl (Fig. 1). which increase manufacturing costs. Oral administration is Ge-132 is thought to improve health as a potent antioxidant still the desired route of administration for most drugs based and enhances the immune response by inducing interferon-g on ease, convenience, pain avoidance and versatility of (IFN-g) and improving natural killer (NK) cell, macrophage formulations, factors contributing to higher patient compli- 31 49,50 and cytotoxic T-cell activity. Ge-132 and novel-related syn- ance. Thus, there is continuing need to develop effective thetic derivatives have demonstrated significant anti-tumour anticancer drugs which are activated at the desired points activity in vitro in cancer cell lines and in vivo animal during gastric passage. 32–37 models of some cancers. The exact anticancer mechan- Developing novel oral formulations, including metal- ism still remains relatively unclear, though induction of based drugs, requires proper characterization of the active IFN-g may be key. Ge-132 may also possess DNA binding pharmaceutical ingredient (API) to determine factors such specificity like cisplatin, to inhibit cancer cell proliferation. as structure, purity, solubility, effect of pH and excipient 39 w There are reported cases of Ge-related toxicity; however, compatibility. Synthetic co-polymers such as Eudragit are these were linked to large amounts of Ge (15–426 g) being commonly used tablet coatings which release APIs in a 21,40 ingested over prolonged periods, up to 36 months. pH-dependent manner in a variety of drug-release profiles 51,52 Studies have narrowed acute and chronic Ge-related toxicity including gastric- and intestinal-dependent solubility. specifically to inorganic germanium dioxide, GeO and In this regard, this research was designed to determine the metallic Ge which can accumulate in the liver, kidney and physicochemical characteristics of Ge-132 and use various 21 41 42 spleen, peripheral nerves, lungs and muscle. Since methods of spectroscopic analysis to determine the purity Ge-132 is also synthesized from GeO , it is possible that of Ge-132 prior to formulation. The main objective of this residual GeO can contaminate formulations claiming to small-scale laboratory study was to develop simple tablets be pure organogermanium. Organogermanium compounds containing known amounts of pure Ge-132 as the API, including Ge-132 have characteristic low toxicity and are using various conventional tabletting excipients. The study readily excreted via the kidney with very low accumulation was also designed with the intention of developing gastro- 40,43 in major organs and tissues. resistant formulations which can withstand the low Most anticancer medicines are administered parenterally stomach pH to prevent or reduce gastric acid degradation, to maximize therapeutic plasma concentrations in the and dissolve in the higher alkaline pH of the lower gastroin- shortest time and to resolve bioavailability issues with testinal tract (GIT) for distribution to the target site. More other formulations. As such, platinum drugs which are importantly, as these tablets contain known amounts of API, they can be easily administered within a convenient and simple oral dosing chemotherapy regimen, incorporating the advantages of oral administration. Materials and Methods Materials Carboxyethylgermanium sesquioxide (Ge-132, 99%) was obtained from Gelest, Inc. (Morrisville, PA, USA). Hydroxypropylmethylcellulose (HPMC) was obtained from The Dow Chemical Company (Midland, MI, USA). Figure 1. Step-wise synthesis of germanium sesquioxide, Ge-132, from w w Eudragit S-100 (ES100) and Eudragit L-100 (EL100) germanium trichloride (germanium chloroform), HGeCl . Adapted from Chang et al. were obtained from Rohm GmbH & Co. KG, Pharma ......................................................................................................................................................................................................................................... 129 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... Polymers (Darmstadt, Germany). Microcrystalline cellulose from 4000 to 530 cm . A second solid sample was analysed (MCC), lactose, deionized water, deuterium water (D O) in an FX Raman Nicolet 9600 NXR spectrometer (4000 to and food colourings (red, green, blue and yellow) were gen- 100 cm ) for comparison. Finally, a Raman analysis of a erously provided by The University of Reading, School of saturated solution of the compound in deionized water was Pharmacy (Reading, Berkshire, UK). Polyplasdone XL-10 performed for further comparison. (Crospovidone) was obtained from ISP (Switzerland) AG. Ultraviolet–visible light spectroscopy Magnesium (Mg) stearate, starch, talc and glycerol were of analytical grade and obtained from Fischer Scientific Ultraviolet–visible (UV–Vis) spectroscopy was recorded on (Loughborough, Leicestershire, UK). a Perkin Elmer Lambda 25 UV–Vis Spectrometer. Stock solutions of Ge-132 (1 mg/ml) were prepared in deionized Methods of analysis of Ge-132 water and spectra recorded from 600 to 190 nm in a quartz cuvette to determine a suitable wavelength for absorp- Solubility testing tion. Serial dilutions were carried out using the stock 5 to 10 mg samples of Ge-132 were dissolved in 25 ml of solutions (0.5, 0.25, 0.20, 0.125, 0.0625 and 0.05 mg/ml) various laboratory solvents (added in 5 ml increments) with and spectra recorded at 212 nm using deionized water as heating in an ultrasonicated bath for 5–30 min. The solvents the reference (blank) to generate a standard calibration were deionized water, 5% NaCl, 5% H SO , 5% HCl, 2 4 curve of absorbance. Finally, a time-dependent scan of a dimethyl sulfoxide (DMSO), methanol, ethanol, diethyl 1mg/ml sample over 15 h was performed to observe any ether and chloroform. Due to time constraints and reagent changes in absorbance over time. availability, testing of Ge-132 solubility in simulated intesti- nal fluid such as phosphate buffer (pH 6.8) was not possible Preparation of uncoated Ge-132 tablets at the time of this research. To determine the maximum Tabletting procedures in this research were limited due to the solubility of Ge-132, 10 mg increments of the compound scale of the study. Uncoated biconvex tablets containing were added to 100 g of hot water (958C) with stirring until Ge-132 as the API were prepared by direct manual com- no further dissolution was possible. pression of dry powder blends containing the API and con- ventional pharmaceutical tabletting excipients. An initial pH testing test formulation (Formulation 1) was prepared primarily to A1 mg/ml stock solution of pure Ge-132 in hot water (958C) determine a suitable bulk mass for dry powders using was prepared and the pH of the solution measured over 30 min lactose (filler) and MCC (compression aid and dry binder). (min) using a SevenEasy Mettler Toledo pH Meter. This formulation was also used to determine compressibility and compression parameters for the dry powder as well as an Melting point determination optimal tablet weight, as the excipients comprised majority The melting points of five samples of pure Ge-132 were of the dry powder. measured using a Stuart SMP10 Melting Point Apparatus. With the exception of Formulation 1, Ge-132 content was maintained conservatively at 5% (250 mg) for all formu- 13 1 C and H nuclear magnetic resonance spectroscopy lations in 5 g bulk powder batches (Table 1). Lactose 13 1 C and proton ( H) nuclear magnetic resonance (NMR) content was kept at 40–50% to increase the bulk powder spectra were recorded at 25.18C (298.1 K) in a Bruker volume. MCC was included at 25–40% to improve com- Avance III 500 MHz spectrometer using D O as a solvent. pression and adhesive properties of the powders. Mg stearate Approximately 1 mg of pure Ge-132 was dissolved in (lubricant) was maintained at 1% to promote powder flow. 5 ml of solvent with gentle heating and analysed to Tablet disintegrants (starch and crospovidone) were main- determine the structure of the compound. tained at 10%. HPMC was added as an additional binder and coating between 4% and 10% of the dry weight. Mass spectrometry The powders were ground in a mortar and sieved in a A 2 mg sample was dissolved in 2 ml of deionized water Fritsch Vibratory Sieve Shaker to achieve a particle size of and analysed by direct injection in a Thermo Fisher 180 mm. Bulk powders were mixed in a rotary Turbula Scientific LTQ Orbitrap XL mass spectrometer over 60 min Type 2C Mixing System for 2.5 min then compressed in a to determine the relative abundance of Ge-132 isotopes in RIVA Minipress MII bench-top eccentric single-punch the sample. tablet press, maintaining the die cavity at a depth of 7 mm. FT-infrared and FT-Raman spectroscopy Preparation of Eudragit dispersions and solutions An FT-infrared (FT-IR) spectrum of a solid sample of ES100 and EL100 aqueous dispersions and organic solutions Ge-132 was recorded on a Perkin Elmer Spectrum 100 were prepared using slight modifications of previous 53–55 FT-IR Spectrometer and analysed within the mid-IR region, research (Table 2). ......................................................................................................................................................................................................................................... 130 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Table 1. Preparation of uncoated Ge-132 tablets using conventional excipients (all weights in grams) Formulation 1 (test) Formulation 2 Formulation 3 Formulation 4 ........................................................................................................................................................................................................................................ API Ge-132 0.5 0.25 0.25 0.25 Filler Lactose 3.75 2.5 2.5 2 Compression aid/dry binder Microcrystalline cellulose (MCC) 1.875 1.25 1.25 2 Lubricant Mg stearate 0.06 0.05 0.05 0.05 Binder/disintegrant Crospovidone – – 0.5 0.5 Starch – 0.5 – – Dry coating Hydroxypropylmethyl cellulose (HPMC) – 0.45 0.5 0.2 Total dry powder weight 6.185 5.0 5.05 5.0 % API content 8% 5% 4.95% 5% Table 2. Preparation of Eudragit S100 and L100 aqueous and 30 min at room temperature. A glycerol–talc mixture and organic formulations (all weights in grams) food colourings (red and yellow) were added to the w w polymer solution and stirring continued for a further 60 min. Eudragit S100 Eudragit L100 ................................................................................................................ Incorporation of aqueous ES100 and EL100 Aqueous Dispersions into dry powders Mass of polymer 10 10 Water 50 50 Due to the small volumes of bulk powder used in this research, we attempted manual film coating of the tablet surfaces; NH OH (1 M) 5 5 however, limitations in the adjustment of droplet size pre- Talc 0.5 0.5 vented uniform coating. As an alternative, bulk dry powders Glycerol 5 5 of Formulation 4 were prepared with ES100 and EL100 Organic solutions aqueous dispersions incorporated into the mixture at Mass of polymer 5 5 20–22% of the dry powder weight, prior to direct com- Ethanol (95%) 75 75 pression (Formulations 5–9) (Table 3). The polymer mixtures Talc 0.25 0.25 were added dropwise with continuous high-speed mixing Glycerol 0.5 0.5 using a magnetic stirring bar to break up agglomerates. Mixing was continued for a total of 20 min until a relatively uniform powder (by sight) was formed. The mixture was left Preparation of ES100 and EL100 aqueous dispersions to dry at room temperature for 60 min, ground and sieved to Granules of each polymer were dispersed in deionized water at obtain particles of 75 mm to enhance powder flow and com- room temperature with continuous high-speed stirring for pressibility. The final powder was reweighed, mixed in a rotary 60 min using an IKA Werke RCT Basic heated magnetic mixer and compressed into tablets with the die cavity adjusted stirrer. To partially neutralize the carboxylic groups of the to 8.5 mm to accommodate more dry powder. polymers, 1 M ammonium hydroxide was added. A separate Quality testing of tablets mixture of talc (glidant) and glycerol (plasticizer) was prepared and stirred for 30 min. The glycerol–talc mixture All formulated tablet batches were subjected to various stan- was gradually added to the polymer dispersion with constant dard quality tests of the British Pharmacopoeia (BP) 2010, stirring. Finally, two drops of food colourings (blue and including consistency of formulation and disintegration green) were added and mixing continued for a further 60 min. testing. Preparation of ES100 and EL100 organic solutions Uniformity of weight (Mass) Granules of each polymer were gradually dispersed into 96% Twenty tablets from each batch were individually weighed ethanol with continuous high-speed stirring for at least on a Mettler AT261 Delta Range scale. Average weights ......................................................................................................................................................................................................................................... 131 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... w w Table 3. Preparation of Ge-132 5% tablets with incorporated Eudragit S100 and Eudragit L100 (all weights in grams) Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 ........................................................................................................................................................................................................................................ Ge-132 0.25 0.25 0.25 0.25 0.25 Lactose 2 2 2 2 2 MCC 2 2 2 2 2 Mg stearate 0.05 0.05 0.05 0.05 0.05 Crospovidone 0.5 0.5 0.5 0.5 0.5 HPMC 0.2 0.2 0.2 0.2 0.2 Eudragit S100 1 – 0.5 0.8 0.35 Eudragit L100 – 1 0.5 0.4 0.7 Final dry powder weight 4.10 4.08 4.14 4.23 4.05 and standard deviation for each batch were calculated and The melting point of the compound proved too high to analysed using BP guidelines to assess batch quality. measure using the laboratory apparatus available (tempera- ture limit of 3508C). At 3308C, Ge-132 decomposed to a Disintegration tests brown solid with no visible droplets forming to indicate 22,57,58 melting. Disintegration testing was performed using a pre-assembled 6-well basket rack in a Copley Erweka ZT42 Disintegration 1 13 56 H and C NMR spectra of Ge-132 Apparatus maintained at 378C. Foreach batch, six randomly The H NMR (D O) spectrum showed two identical proton selected tablets were placed into the basket-rack and covered 2 with a 20 mm plastic disc. The basket was lowered into environments at 1.55 and 2.65 ppm, and the C NMR (D O) spectrum showed three distinct carbon environments 700 ml of immersion fluid and the apparatus operated for 2 at 178, 27 and 12.5 ppm. The peaks in both spectra were 15 min. The immersion fluids were 0.1 M HCl (pH 1.36) and deionized water (pH 7.4). The disintegration time was consistent with the proposed molecular structure and 57,58 monomeric formula of Ge-132. recorded as the time for the last of the six tablets to fully disintegrate. Disintegration tests were performed twice for Mass spectrum of Ge-132 each formulation in each of the immersion fluids. Hydrolysis of the compound resulted in numerous fragments Uniformity of content of varying mass presented in the full mass spectrum of Ge-132 (Fig. 2). The complex spectrum shows the relative Uniformity of content testing was carried out on abundances of distinct Ge-containing isotope clusters with Formulations 2–9 according to BP guidelines. Ten tablets splitting patterns very similar to those previously were individually weighed, crushed and dissolved in 59,60 reported. Additionally, these clusters occur in suggested 1000 ml of hot deionized water, then filtered to remove overlapping regions as indicated. These clusters indicate undissolved excipients. Two millilitres of samples were either incomplete fragmentation of the solid structure or analysed by UV–Vis (212 nm) and the absorbances used to formation of new linked Ge-132 units, possibly cyclic and calculate the content of Ge-132 in the tablets (against the 59–61 linear. standard calibration curve). The acceptance values (AVs) for deviations were calculated according to BP guidelines. FT-IR and FT-Raman spectroscopy of Ge-132 The FT-IR spectrum of the solid compound shows character- Results istic carboxylic O–H bond and C¼O bond vibrational peaks at 3300.00–2700.00 and 1687.55 cm , respectively Analysis of Ge-132 (Fig. 3). The peaks at 1410.00 and 1236.65 cm indicate Solubility, pH and melting point asymmetrical and symmetrical C–H bond vibrations of the Ge-132 fully dissolved in water forming an acidic solution of alkyl chains. Most notable in this mid-IR spectrum are the pH 3.06–3.12, and fully dissolved in common aqueous prominent characteristic sesquioxide peaks at 900.51 and laboratory solvents including 5% NaCl, 5% H SO and 800.04 cm , consistent with previous studies of pure 2 4 36,37 5% HCl. The compound was insoluble in common organic Ge-132 and its derivatives, confirming the –O–Ge– laboratory solvents (methanol, ethanol, diethyl ether and O–Ge-type network. chloroform) and only partially soluble in DMSO with The comparative FT-Raman spectrum of solid Ge-132 heating. (Fig. 4, top) shows more distinct carboxylic O–H bond ......................................................................................................................................................................................................................................... 132 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Figure 2. Full MS spectrum of pure Ge-132 showing suggested overlapping regions and % relative abundances of polymeric ions of Ge-132 in aqueous solution. Overlapping regions result from the polyisotopic nature of elemental Ge as well as loss or addition of neutral species (see text). A, monomeric ions; B, dimeric ions; C, region consisting of more complex trimeric, tetrametric, pentameric and hexameric ions. 21 21 Figure 3. FT-IR spectrum of Ge-132 solid showing % transmittance of O–H bonds (3300.00–2700.00 cm ) and C¼O bonds (1687.55 cm ) of the car- boxylic groups, –COOH. The peaks at 1410.00 and 1236.65 cm indicate C–H bond vibrations, and the characteristic sesquioxide peaks are visible at 900.51 and 800.04 cm . Figure 4. Comparative FT-Raman spectra of Ge-132 solid (top) and Ge-132 aqueous solution (bottom). Peaks in Ge-132 solid at 2932.90 (–COOH), 900.26 21 21 (sesquioxide) and 445.57 cm (–Ge–C–) are hydrolysed in solution to the broader 3361.96, 890.85 and 472.03 cm peaks, respectively. vibrations at 2932.90 cm as well as the characteristic aqueous Ge-132 in Fig. 4 (bottom) shows the vibrational sesquioxide peak at 900.26 cm . New peaks were frequencies of the hydrolysis products of Ge-132 with observed at 688.02 and 445.57 cm , the former peak indi- broader peaks observed at 3361.96, 1643.06 and 62 21 cating Ge–C bond vibrations. The Raman spectrum of 1437.44 cm . ......................................................................................................................................................................................................................................... 133 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... incorporated into the bulk powder had lower disintegration times (1 min 13 s to 1 min 36 s), though this was due to the smaller particle size. Uniformity of content test Mean absorbances at 212 nm ranged from 0.114 to 0.913. Using our calibration curve, we converted the absorbances into mean API content with the following results: Figure 5. UV–Vis calibration curve for Ge-132 at 212 nm for 0.00, 0.10, Formulation 2, 4.95%; Formulation 3, 4.68%; Formulation 0.20, 0.25, 0.50 and 1.0 mg/ml solutions. 4, 4.80%; Formulation 5, 5.02%; Formulation 6, 5.18%; Formulation 7, 2.45%; Formulation 8, 3.73%; and UV–Vis spectroscopy of Ge-132 Formulation 9, 0.65%. The mean % Ge-132 content for each formulation was then analysed using the BP formula for Between 215 and 210 nm, the serial dilutions we prepared calculating AV: showed consistent absorption within these wavelengths as well as relatively minimal variation in the gradient between jM  Xjþ ks the serial dilutions. For this reason, 212 nm was chosen as a suitable wavelength to study Ge-132 UV absorbance and where M is the reference value from the mean API content as a a resulting calibration curve produced (Fig. 5). Over 15 h, percentage of the label claim; X the mean content; k the a1mg/ml solution of Ge-132 had constant absorbance acceptability constant or tolerance interval (2.4 for 10 (A¼0.287) at 212 nm. tablets) and, s the standard deviation. An AV of 15 (termed L1) denotes a successful Analysis of tablet formulations formulation. Tablets of Formulations 1–5 measured 6 mm in diameter The AVs were calculated as 0.01, 4.87, 2.51, 0.00, 2.13, with a thickness of 2 mm, whereas Formulations 6–9 were 49.71, 24.49 and 86.07 for Formulations 2–9, respectively. slightly thicker at 4–5 mm with a similar surface diameter. Formulation 4 containing a 1:1 lactose:MCC mixture yielded a powder with better compressibility than Formulations 1–3 which had a 2:1 (lactose:MCC) mixture. Discussion This formulation was therefore chosen for further develop- Solubility, pH and melting (decomposition) point ment in Formulations 5–9. Ge-132, a white, crystalline powder, exists as a complex, infi- Uniformity of weight (Mass) test nite crystal network of O–Ge–O bonds with the basic Tablets of Formulations 2–9 passed BP test of Uniformity of monomeric formula O (GeCH CH COOH) (Fig. 6A). 3 2 2 2 Weight (Mass) with no sample deviating above or below Ge-132 fully dissolves in water and equilibrates to the trihy- the mean respective batch weight by .7.5%. Tablets in droxyl, germane triol, (OH) GeCH CH COOH (Fig. 6B), 3 2 2 Formulations 2–4 had mean weights of 116.45+ 3.06, forming an acidic solution (pH 3.06–3.12). Across all 110.33+ 4.06 and 109.38+ 3.75 mg, respectively. As tested solvents, water provided the best dissolution medium expected, tablets in Formulations 5–9 were heavier due to with the saturation solubility of Ge-132 determined to be incorporation of aqueous Eudragit . Mean weights were 1.28 g per 100 g of water. We also observed that even 123.40+ 3.15 mg (Formulation 5), 148.11+ 2.13 mg after cooling and over a period of 5 days, the compound (Formulation 6), 133.26+ 1.79 mg (Formulation 7), remained in solution and did not precipitate out. Stability 131.75+ 3.49 mg (Formulation 8) and 134.01+ 2.89 mg testing of the compound in solution over longer periods of (Formulation 9). time is an important aspect of future research which will provide further data on the viability of Ge-132 as a novel Tablet disintegration tests anticancer drug. Disintegration times for all formulations were determined in The melting (decomposition) temperature we measured both acidic and alkaline media. Tablets of Formulation 1 at 3308C is consistent with previously reported literature fully disintegrated within 13 s in both solutions which was and also expected from the known melting temperature expected, considering the powder did not contain significant of elemental Ge of 937.48C. The high melting amounts of binder. Formulations 2–4 had longer disinte- (decomposition) point is attributable to the crystal gration times, between 2 min 55 s in acidic pH and 3 min structure resulting from complex polymerization of the 10 s in alkaline pH. Formulations 5–9 with Eudragit monomeric unit. ......................................................................................................................................................................................................................................... 134 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Figure 6. (A) Structural representation of the basic monomeric unit of solid Ge-132, where R ¼ CH CH COOH, and n denotes the degree of polymerization 2 2 (adapted from Sawai et al. ). (B) Formation of germane triol (trihydroxylgermane) from hydrolysis of solid Ge-132. Figure 7. Proposed structures of some cyclic ions identified from peaks in the MS analysis of pure Ge-132 in aqueous solution, showing monomeric (A), dimeric (B and C) and trimeric (D and E) ion fragments of Ge-132 ( Ge) with predicted and actual m/z values. The loss of neutral species alters the shape of the ions, for example, structure C results from the loss of neutral CO and CH ¼CH from structure B (adapted from Wie et al. ). 2 2 2 NMR spectroscopy number of Ge atoms, and vary in mass depending on the 1 13 constituent Ge isotopes within each ion. H and C NMR were performed to confirm the structure of The smallest linear units are possibly the monomeric Ge-132. D O was chosen as the analysis solvent due to the þ þ ions (OH) Ge , (OH) GeCH CH and (OH) GeCH CH 3 3 2 2 3 2 2 favourable solubility in water. In the H NMR spectrum, COOH with calculated masses of m/z 125, 153 and 199, the triplet peaks at 1.55 and 2.65 ppm indicate proton respectively. These ions are present in our MS spectrum at pairs in the –Ge–CH –CH – and –CH –CH –COOH– 2 2 2 2 m/z 123.1, 151.1 and 193. Similarly, the basic cyclic units regions, respectively. These proton environments are dis- can be any of several different forms (Fig. 7A–E) ranging tinguishable by the proximity of the latter protons to the in mass from m/z 181 to 480. The peaks of these units are electronegative carboxyl oxygens, which shifts them further present in our spectrum within the monomeric, dimeric downfield on the chemical shift (d) scale. The d values are and trimeric regions from m/z 181 to 478.3. consistent with previously reported values of 1.61 and Another complication of interpreting the MS of Ge-132 is 2.69 ppm. fragmentation from addition or loss of neutral species—such The peaks presented in the C NMR spectrum were also as H O, CO ,CH ¼CH and GeO—can change the shape 2 2 2 2 consistent with the expected molecular structure and 57,58 and isomeric nature of the ion. For example, the prominent structural data for pure Ge-132 previously reported. peak at m/z 213.1 may be the linear dimeric fragment These peaks confirm the presence of carbons as follows: (OH) GeOGe , or the cyclic dimeric fragment in Fig. 7C 178 ppm, Ge–CH –CH –COOH; 27 ppm, Ge–CH – 2 2 2 resulting from loss of CO and CH ¼CH . Similarly, the 2 2 2 CH –COOH and, 12.5 ppm, Ge–CH –CH –COOH. 2 2 2 peak at m/z 193.0 could be the linear monomeric ion These d values are comparable to those previously as (OH) GeCH CH COOH , or possibly the linear dimeric 3 2 2 2 181.30, 28.43 and 15.68 ppm. ion HOGeOGeOH . The spectrum is further complicated by the polyisotopic nature of Ge where each isotope pro- MS spectrometry duces its own characteristic splitting pattern. This means Elemental Ge exists in five known stable isotopes— Ge that any combination of isotopes can be present in a frag- 72 73 74 (20.5%), Ge (27.4%), Ge (7.8%), Ge (36.5%) and ment causing overlapping peaks which may not be visible 76 61 Ge (7.8%). This polyisotopic nature produces a in the spectrum or identifiable in Fig. 2. complex MS spectrum with significant overlapping regions For the purposes of interpreting our spectrum, all masses consisting of different ions. Furthermore, these ions are poly- were calculated using the most abundant isotope, Ge, meric (monomeric, dimeric, trimeric, etc.) depending on the which accounts for the differences in m/z values between ......................................................................................................................................................................................................................................... 135 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... our calculated and measured masses. Further detailed MS Ge-132 tablet formulations analysis beyond the scope of this research is required to Preparation of most tablet formulations involves multistep accurately determine the exact isotopic composition of the processes including dry granulation, wet granulation and compound and conclusively ascertain the identity of the frag- fluid bed drying, prior to compression. However, due to ments. The spectrum however remains significant as it the small-scale nature of this study, preparation methods further confirms the purity and solubility products of our were limited to direct compression. All tablets were biconvex API and, most importantly, the absence of fragments of and produced by manual rotation of a single punch press. BP inorganic GeO . tests of resistance to crushing and tablet friability were not performed at the time due to technical failure of the available IR spectroscopy equipment. The higher loss in bulk powder weight prior to For additional confirmation of the purity and constituent compression in Formulations 5–9 can be attributed to the bonds in Ge-132, both FT-IR spectroscopy and FT-Raman extra processing steps (mixing, grinding, sieving and choos- spectroscopy were performed. Inorganic GeO absorbs ing a fraction of 75 mm) used to incorporate Eudragit strongest at 850 cm and produces a distinct characteristic into the bulk powder. peak in both FT-IR and Raman spectra. For identification Two interesting observations were made: first, despite pre- of functional groups which may not be visible in the FT-IR paring Formulation 5 with the reduced particle size and spectrum, FT-Raman spectroscopy was performed to detect increased die cavity depth, the tablets measured 6  2 mm, weaker vibrational frequencies in the far-IR region below similar to Formulations 1–4. It is possible that ES100 may 600 cm . Most notable is the peak observed at have altered the powder characteristics or the processing 445.57 cm ; however, without further analysis, the identity may have changed the flow and compaction properties of of this peak remains unknown and to date, there are no the powder, although the exact reason requires further inves- reported studies identifying this peak. Most important for tigation. Second, tablets of Formulation 6 were notably this research was conclusive confirmation of the absence of heavier (mean weight 148.11 mg) and thicker (5 mm), toxic, inorganic GeO (at 850 cm ). despite the same particle size and die cavity depth as The broader peaks at 3361.96, 1643.06 and Formulations 5, 7, 8 and 9. This may also be attributed to 1437.44 cm in the Raman spectrum of aqueous Ge-132 the constituent polymer (EL100) or the powder processing denote bond vibrations from –COOH and alkyl chain and emphasizes the need for further formulation studies. hydrolysis consistent with the MS fragments presented The fast disintegration times across all formulations are above. Of particular interest is the absence of the sesquioxide also attributable to the tablet manufacturing process using peaks at 800 and 900 cm previously seen in the FT-IR and dry granulation and direct manual compression of the dry Raman spectra of solid Ge-132, indicating hydrolysis of the powders. Wet granulation with a liquid binder to sufficiently crystal network and formation of newer –O–Ge–O– links wet the dry powders would be a more effective method of as seen in our MS. Finally, the peak between 890.86 and producing homogenous granules of API and excipients 708.49 cm in Fig. 4 (bottom) has been identified as charac- with better compaction properties. However, the disinte- teristic Ge–H bond vibrations within the trihydroxylger- gration times reported here provide a baseline for further for- mane, H–Ge–(OH) . The absence of most peaks mulation studies using alternative granulation methods. between 1400.00 and 601.37 cm is further evidence of Increasing the tablet dimensions by modifying the tabletting the formation of new hydrolysis products and, as seen machine parameters may also improve disintegration times previously, the GeO peak at 850 cm remains absent. as demonstrated by Obeidat et al. but should be made with consideration of the purpose of any oral formulation, UV–Vis spectroscopy bearing in mind various patient factors. Various wavelengths for UV–Vis spectroscopy (in water) There are various possibilities why Formulations 7–9 have been reported for Ge-132, from 190 to 210 nm. failed content uniformity testing, determined by the Because these wavelengths occur at the lower detection calculated AV. The polymer combinations within each for- limit of our spectrometer (190 nm), a series of scans from mulation may have altered the API characteristics; alterna- 220 to 190 nm were performed on dilute Ge-132 solutions. tively, the extra mixing step and decreased particle size At 212 nm, the absorbance series of the serial dilutions after polymer incorporation may have resulted in particle were most linear and this was chosen as a suitable wave- segregation which decreases powder quality and affects the length for this research. The 15 h (900 min) time-dependent distribution of API particles. Interestingly, a previous study UV–Vis absorbance at 212 nm showed constant absorbance using ES100–EL100 combinations as tablet coatings for (A ¼ 0.287), indicating no further decomposition of the the acidic drug mesalazine determined that a higher compound in solution and the overall stability of the EL100:ES100 ratio produced a more favourable drug-release hydrolysis products. profile when the ratio was kept above 4:1. ......................................................................................................................................................................................................................................... 136 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Further large-scale studies using fluidized spray coating further large-scale formulation studies to assess the compat- will provide useful data on the compatibility of polymer ibility and suitability of polymers coatings such as Eudragit combinations with Ge-132 to formulate a novel gastro- with Ge-132, taking into account the acidic nature and solu- resistant anticancer treatment. The formulation studies bility characteristics of Ge-132. Additionally, further must also consider the reported solubility of Ge-132 in research should also appropriately determine Ge-132 solubi- water, the primary solvent used to prepare the polymer lity in simulated intestinal fluid or phosphate buffer (pH 6.8) suspensions. to mimic the natural passage of oral formulations through the GIT. Eudragit tablet formulations Finally, with advancements in nanotechnology and A range of Eudragit co-polymers are widely used to targeted drug delivery, future research might also focus on produce various modified-release profiles determined by the development of nano-formulations of Ge-132 beyond relative ratio of methacrylic acid to methyl methacrylate conventional gastro-resistant tablets. This research could ester within each polymer. In ES100, this ratio is 1:2 (acid:e- evaluate the compatibility of Ge-132 with various nanocar- ster), making it selectively soluble at pH .7.0 around the riers and formulation of the compound into novel nanopar- ileum and colon. The constituent ratio in EL100 is 1:1, ticulate systems such as Ge-132-polymer conjugates, making it soluble at a slightly lower pH (6–7) and useful liposomes and micelles. 52,53 for drugs targeting the jejunum. ES100 and EL100 have very similar physicochemical characteristics and for 51 Acknowledgements this reason were prepared in the same way. Dispersion of dry Eudragit powders in water and I would like to thank my project supervisors, Dr Katja addition of ammonia (1 M) produced a milky latex. This Stohfeldt-Venables and Dr Clare Rawlinson Malone, for consistency allowed us to gradually incorporate the their invaluable mentoring, guidance and supervision Eudragit dispersions dropwise into our dry powders as an during this exciting and challenging project. I would also alternative to wet granulation. In contrast, ES100 and like to acknowledge the Reading School of Pharmacy techni- EL100 formed glossy and highly viscous solutions in cal staff for the time they dedicated to providing support and ethanol which prevented dropwise addition. Moreover, use guidance throughout this study. of organic coatings is steadily declining in the pharma- ceutical industry due to environmental and health con- Funding cerns. Despite variable results from Formulations 5–9, future research of gastro-resistant Ge-132 formulations Funding and support for this project was provided by the should aim to maintain the particle size between 75 and School of Pharmacy, University of Reading, as part of the 180 mm, in addition to analysing the actual effect of Undergraduate Final Year Project module. polymers such as Eudragit on Ge-132. Author biography Conclusion and future studies S.M.O. graduated in July 2010 with an undergraduate The use of metals in medicine has significantly increased, Masters degree in Pharmacy from School of Pharmacy, particularly in developing novel anticancer therapies. The University of Reading, UK. She is currently undertaking anti-tumour activity of Ge-132 has been reported in numer- 1 year Pre-registration Pharmacist training in hospital phar- ous studies warranting further development as a novel macy. Special interests include paediatric pharmacy and anti-cancer drug. Despite the small scale of this study, we formulation of drugs for paediatric administration. were able to determine the purity of Ge-132 using Therapeutics areas of particular interest include cardiology, common spectroscopic techniques which are also efficient cancer and HIV. She is hoping to pursue a PhD in pharma- methods of detecting the presence of toxic GeO . ceutical formulation. Nearly, all anticancer drugs are administered parenterally, despite the oral route being the most popular route of admin- istration due to its ease, convenience and general patient References acceptability. Ge-132, a freely water-soluble compound, is 1. 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Analysis of Ge-132 and development of a simple oral anticancer formulation

Bioscience Horizons , Volume 4 (2) – Jun 6, 2011

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Volume 4 † Number 2 † June 2011 10.1093/biohorizons/hzr015 Advance Access publication 6 May 2011 ......................................................................................................................................................................................................................................... Research article Analysis of Ge-132 and development of a simple oral anticancer formulation Sara M. Ogwapit* School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AH, UK. * Corresponding author: Email: s.o.malinga@gmail.com Supervisors: Dr Katja Strohfeldt-Venables and Dr Clare Rawlinson Malone, School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, UK. ........................................................................................................................................................................................................................................ The anticancer activity of synthetic organogermanium, b-or bis-carboxyethylgermanium sesquioxide (Ge-132), has been demonstrated in several cancer cell models and human studies. Ge-132 increases pro-inflammatory responses by enhancing interferon-g (IFN-g), natural killer cell and T-cell activity, and is significantly less toxic than other widely used metal-based anticancer drugs such as cisplatin. In this small-scale laboratory study, we effectively assessed the physicochemical characteristics and purity of Ge-132, our main objective being to develop a novel oral anticancer formulation, using conventional tabletting excipients which do not alter the chemistry of Ge-132. We determined that solid Ge-132 decomposes at 3308C; is virtually insoluble in most common organic solvents; and readily 1 13 dissolves in water (saturation solubility 1.28 g/100 ml) to form germane triol (pH 3.06–3.12). H and C nuclear magnetic resonance spectroscopy confirmed the structure of our compound showing two identical proton environments at 1.55 and 2.65 ppm (triplets) and three distinct carbon environments at 178.31, 27.37 and 12.93 ppm. The mass spectrum indicated the formation of numerous complex ion fragments with masses ranging from m/z 123.1 to m/z 478.3. FT-infrared and FT-Raman spectra showed characteristic sesquioxide 21 21 peaks at 900.51, 900.26 and 800.04 cm and, most importantly, confirmed the absence of toxic, inorganic GeO , at 850 cm . While parenteral formulations exist for many anticancer medicines, here we successfully developed uncoated tablets containing Ge-132 (5% w/w) by manual direct compression (powder particle size 180 mm). The tablets passed British Pharmacopoeia (BP) content uni- formity testing (Ultraviolet–visible, 212 nm), and BP disintegration testing in both acidic and basic media, disintegrating between 2 min 55 s and 3 min 10 s, respectively. We prepared gastro-resistant formulations using Eudragit ; however, these failed content uniformity tests and had lower disintegration times (1 min 36 s), indicating that compatibility of polymers with Ge-132 requires further investi- gation. The results presented here support further larger-scale research on Ge-132 as a novel metal-based oral anticancer drug which can be conveniently administered alone or included within a chemotherapy regimen. Future formulation studies on Ge-132 could focus on compatibility assessments with nano-formulations in keeping with current advancements in metal-based anticancer therapies. Key words: Ge-132, spectral analysis, anticancer, oral formulation. Submitted September 2010; accepted February 2011 ........................................................................................................................................................................................................................................ platinum (II) and platinum (IV) complexes which inhibit Introduction 5,6 cell division. Cisplatin is one of the most successful metal- Despite advancements in cancer research and drug delivery, based anticancer drugs effective against a diverse range of many cancers still do not have adequate targeted treatments. tumour types, though it is associated with significant renal Modern anticancer therapies show improved clinical efficacy toxicity and induced or acquired resistance. The search for and specificity towards cancer cells; however, their success is novel anticancer drugs continues and other metals have 7,9–20 limited by the development of resistance and numerous side been investigated for their anticancer potential, includ- 2–4 effects, complicating their use in chemotherapy regimens. ing germanium (Ge), a naturally occurring metalloid found Metal-based anticancer therapy arose from the discovery of in soil. ......................................................................................................................................................................................................................................... The Author 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited 128 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Ge is a constituent of many plants, including garlic and aquated and activated within the cell cytoplasm require ginseng root, and may play a role in photosynthetic, self- sufficient protection to bypass healthy cells and target defence and metabolic processes. The daily intake of cancer cells. Although there are several novel formulations 44–46 elemental Ge from food is estimated between 367 and of successful platinum-based drugs, there are limited 3700 mg and both organic and inorganic Ge compounds formulation studies of other metal-based anticancer drugs have been used for many years as a dietary supplement in (including organogermanium compounds) into viable drug doses of 15 mg–1 g per day. Various synthetic organic delivery forms as most organometallic chemistry does not Ge compounds have been investigated for their therapeutic incorporate formulation science. efficacy in osteoporosis, malaria, hypertension, cancer and Parenteral drug administration is limited to hospitals or 24–29 AIDS. One of the most widely researched is the sesqui- specialized treatment centres where patients rely on trained oxide, b-or bis-carboxyethylgermanium sesquioxide, also professionals using complex equipment. Additionally, par- known as Ge-132, an organogermanium synthesized from enteral formulations require sterile manufacturing conditions 30 48 the chloroform, HGeCl (Fig. 1). which increase manufacturing costs. Oral administration is Ge-132 is thought to improve health as a potent antioxidant still the desired route of administration for most drugs based and enhances the immune response by inducing interferon-g on ease, convenience, pain avoidance and versatility of (IFN-g) and improving natural killer (NK) cell, macrophage formulations, factors contributing to higher patient compli- 31 49,50 and cytotoxic T-cell activity. Ge-132 and novel-related syn- ance. Thus, there is continuing need to develop effective thetic derivatives have demonstrated significant anti-tumour anticancer drugs which are activated at the desired points activity in vitro in cancer cell lines and in vivo animal during gastric passage. 32–37 models of some cancers. The exact anticancer mechan- Developing novel oral formulations, including metal- ism still remains relatively unclear, though induction of based drugs, requires proper characterization of the active IFN-g may be key. Ge-132 may also possess DNA binding pharmaceutical ingredient (API) to determine factors such specificity like cisplatin, to inhibit cancer cell proliferation. as structure, purity, solubility, effect of pH and excipient 39 w There are reported cases of Ge-related toxicity; however, compatibility. Synthetic co-polymers such as Eudragit are these were linked to large amounts of Ge (15–426 g) being commonly used tablet coatings which release APIs in a 21,40 ingested over prolonged periods, up to 36 months. pH-dependent manner in a variety of drug-release profiles 51,52 Studies have narrowed acute and chronic Ge-related toxicity including gastric- and intestinal-dependent solubility. specifically to inorganic germanium dioxide, GeO and In this regard, this research was designed to determine the metallic Ge which can accumulate in the liver, kidney and physicochemical characteristics of Ge-132 and use various 21 41 42 spleen, peripheral nerves, lungs and muscle. Since methods of spectroscopic analysis to determine the purity Ge-132 is also synthesized from GeO , it is possible that of Ge-132 prior to formulation. The main objective of this residual GeO can contaminate formulations claiming to small-scale laboratory study was to develop simple tablets be pure organogermanium. Organogermanium compounds containing known amounts of pure Ge-132 as the API, including Ge-132 have characteristic low toxicity and are using various conventional tabletting excipients. The study readily excreted via the kidney with very low accumulation was also designed with the intention of developing gastro- 40,43 in major organs and tissues. resistant formulations which can withstand the low Most anticancer medicines are administered parenterally stomach pH to prevent or reduce gastric acid degradation, to maximize therapeutic plasma concentrations in the and dissolve in the higher alkaline pH of the lower gastroin- shortest time and to resolve bioavailability issues with testinal tract (GIT) for distribution to the target site. More other formulations. As such, platinum drugs which are importantly, as these tablets contain known amounts of API, they can be easily administered within a convenient and simple oral dosing chemotherapy regimen, incorporating the advantages of oral administration. Materials and Methods Materials Carboxyethylgermanium sesquioxide (Ge-132, 99%) was obtained from Gelest, Inc. (Morrisville, PA, USA). Hydroxypropylmethylcellulose (HPMC) was obtained from The Dow Chemical Company (Midland, MI, USA). Figure 1. Step-wise synthesis of germanium sesquioxide, Ge-132, from w w Eudragit S-100 (ES100) and Eudragit L-100 (EL100) germanium trichloride (germanium chloroform), HGeCl . Adapted from Chang et al. were obtained from Rohm GmbH & Co. KG, Pharma ......................................................................................................................................................................................................................................... 129 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... Polymers (Darmstadt, Germany). Microcrystalline cellulose from 4000 to 530 cm . A second solid sample was analysed (MCC), lactose, deionized water, deuterium water (D O) in an FX Raman Nicolet 9600 NXR spectrometer (4000 to and food colourings (red, green, blue and yellow) were gen- 100 cm ) for comparison. Finally, a Raman analysis of a erously provided by The University of Reading, School of saturated solution of the compound in deionized water was Pharmacy (Reading, Berkshire, UK). Polyplasdone XL-10 performed for further comparison. (Crospovidone) was obtained from ISP (Switzerland) AG. Ultraviolet–visible light spectroscopy Magnesium (Mg) stearate, starch, talc and glycerol were of analytical grade and obtained from Fischer Scientific Ultraviolet–visible (UV–Vis) spectroscopy was recorded on (Loughborough, Leicestershire, UK). a Perkin Elmer Lambda 25 UV–Vis Spectrometer. Stock solutions of Ge-132 (1 mg/ml) were prepared in deionized Methods of analysis of Ge-132 water and spectra recorded from 600 to 190 nm in a quartz cuvette to determine a suitable wavelength for absorp- Solubility testing tion. Serial dilutions were carried out using the stock 5 to 10 mg samples of Ge-132 were dissolved in 25 ml of solutions (0.5, 0.25, 0.20, 0.125, 0.0625 and 0.05 mg/ml) various laboratory solvents (added in 5 ml increments) with and spectra recorded at 212 nm using deionized water as heating in an ultrasonicated bath for 5–30 min. The solvents the reference (blank) to generate a standard calibration were deionized water, 5% NaCl, 5% H SO , 5% HCl, 2 4 curve of absorbance. Finally, a time-dependent scan of a dimethyl sulfoxide (DMSO), methanol, ethanol, diethyl 1mg/ml sample over 15 h was performed to observe any ether and chloroform. Due to time constraints and reagent changes in absorbance over time. availability, testing of Ge-132 solubility in simulated intesti- nal fluid such as phosphate buffer (pH 6.8) was not possible Preparation of uncoated Ge-132 tablets at the time of this research. To determine the maximum Tabletting procedures in this research were limited due to the solubility of Ge-132, 10 mg increments of the compound scale of the study. Uncoated biconvex tablets containing were added to 100 g of hot water (958C) with stirring until Ge-132 as the API were prepared by direct manual com- no further dissolution was possible. pression of dry powder blends containing the API and con- ventional pharmaceutical tabletting excipients. An initial pH testing test formulation (Formulation 1) was prepared primarily to A1 mg/ml stock solution of pure Ge-132 in hot water (958C) determine a suitable bulk mass for dry powders using was prepared and the pH of the solution measured over 30 min lactose (filler) and MCC (compression aid and dry binder). (min) using a SevenEasy Mettler Toledo pH Meter. This formulation was also used to determine compressibility and compression parameters for the dry powder as well as an Melting point determination optimal tablet weight, as the excipients comprised majority The melting points of five samples of pure Ge-132 were of the dry powder. measured using a Stuart SMP10 Melting Point Apparatus. With the exception of Formulation 1, Ge-132 content was maintained conservatively at 5% (250 mg) for all formu- 13 1 C and H nuclear magnetic resonance spectroscopy lations in 5 g bulk powder batches (Table 1). Lactose 13 1 C and proton ( H) nuclear magnetic resonance (NMR) content was kept at 40–50% to increase the bulk powder spectra were recorded at 25.18C (298.1 K) in a Bruker volume. MCC was included at 25–40% to improve com- Avance III 500 MHz spectrometer using D O as a solvent. pression and adhesive properties of the powders. Mg stearate Approximately 1 mg of pure Ge-132 was dissolved in (lubricant) was maintained at 1% to promote powder flow. 5 ml of solvent with gentle heating and analysed to Tablet disintegrants (starch and crospovidone) were main- determine the structure of the compound. tained at 10%. HPMC was added as an additional binder and coating between 4% and 10% of the dry weight. Mass spectrometry The powders were ground in a mortar and sieved in a A 2 mg sample was dissolved in 2 ml of deionized water Fritsch Vibratory Sieve Shaker to achieve a particle size of and analysed by direct injection in a Thermo Fisher 180 mm. Bulk powders were mixed in a rotary Turbula Scientific LTQ Orbitrap XL mass spectrometer over 60 min Type 2C Mixing System for 2.5 min then compressed in a to determine the relative abundance of Ge-132 isotopes in RIVA Minipress MII bench-top eccentric single-punch the sample. tablet press, maintaining the die cavity at a depth of 7 mm. FT-infrared and FT-Raman spectroscopy Preparation of Eudragit dispersions and solutions An FT-infrared (FT-IR) spectrum of a solid sample of ES100 and EL100 aqueous dispersions and organic solutions Ge-132 was recorded on a Perkin Elmer Spectrum 100 were prepared using slight modifications of previous 53–55 FT-IR Spectrometer and analysed within the mid-IR region, research (Table 2). ......................................................................................................................................................................................................................................... 130 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Table 1. Preparation of uncoated Ge-132 tablets using conventional excipients (all weights in grams) Formulation 1 (test) Formulation 2 Formulation 3 Formulation 4 ........................................................................................................................................................................................................................................ API Ge-132 0.5 0.25 0.25 0.25 Filler Lactose 3.75 2.5 2.5 2 Compression aid/dry binder Microcrystalline cellulose (MCC) 1.875 1.25 1.25 2 Lubricant Mg stearate 0.06 0.05 0.05 0.05 Binder/disintegrant Crospovidone – – 0.5 0.5 Starch – 0.5 – – Dry coating Hydroxypropylmethyl cellulose (HPMC) – 0.45 0.5 0.2 Total dry powder weight 6.185 5.0 5.05 5.0 % API content 8% 5% 4.95% 5% Table 2. Preparation of Eudragit S100 and L100 aqueous and 30 min at room temperature. A glycerol–talc mixture and organic formulations (all weights in grams) food colourings (red and yellow) were added to the w w polymer solution and stirring continued for a further 60 min. Eudragit S100 Eudragit L100 ................................................................................................................ Incorporation of aqueous ES100 and EL100 Aqueous Dispersions into dry powders Mass of polymer 10 10 Water 50 50 Due to the small volumes of bulk powder used in this research, we attempted manual film coating of the tablet surfaces; NH OH (1 M) 5 5 however, limitations in the adjustment of droplet size pre- Talc 0.5 0.5 vented uniform coating. As an alternative, bulk dry powders Glycerol 5 5 of Formulation 4 were prepared with ES100 and EL100 Organic solutions aqueous dispersions incorporated into the mixture at Mass of polymer 5 5 20–22% of the dry powder weight, prior to direct com- Ethanol (95%) 75 75 pression (Formulations 5–9) (Table 3). The polymer mixtures Talc 0.25 0.25 were added dropwise with continuous high-speed mixing Glycerol 0.5 0.5 using a magnetic stirring bar to break up agglomerates. Mixing was continued for a total of 20 min until a relatively uniform powder (by sight) was formed. The mixture was left Preparation of ES100 and EL100 aqueous dispersions to dry at room temperature for 60 min, ground and sieved to Granules of each polymer were dispersed in deionized water at obtain particles of 75 mm to enhance powder flow and com- room temperature with continuous high-speed stirring for pressibility. The final powder was reweighed, mixed in a rotary 60 min using an IKA Werke RCT Basic heated magnetic mixer and compressed into tablets with the die cavity adjusted stirrer. To partially neutralize the carboxylic groups of the to 8.5 mm to accommodate more dry powder. polymers, 1 M ammonium hydroxide was added. A separate Quality testing of tablets mixture of talc (glidant) and glycerol (plasticizer) was prepared and stirred for 30 min. The glycerol–talc mixture All formulated tablet batches were subjected to various stan- was gradually added to the polymer dispersion with constant dard quality tests of the British Pharmacopoeia (BP) 2010, stirring. Finally, two drops of food colourings (blue and including consistency of formulation and disintegration green) were added and mixing continued for a further 60 min. testing. Preparation of ES100 and EL100 organic solutions Uniformity of weight (Mass) Granules of each polymer were gradually dispersed into 96% Twenty tablets from each batch were individually weighed ethanol with continuous high-speed stirring for at least on a Mettler AT261 Delta Range scale. Average weights ......................................................................................................................................................................................................................................... 131 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... w w Table 3. Preparation of Ge-132 5% tablets with incorporated Eudragit S100 and Eudragit L100 (all weights in grams) Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 ........................................................................................................................................................................................................................................ Ge-132 0.25 0.25 0.25 0.25 0.25 Lactose 2 2 2 2 2 MCC 2 2 2 2 2 Mg stearate 0.05 0.05 0.05 0.05 0.05 Crospovidone 0.5 0.5 0.5 0.5 0.5 HPMC 0.2 0.2 0.2 0.2 0.2 Eudragit S100 1 – 0.5 0.8 0.35 Eudragit L100 – 1 0.5 0.4 0.7 Final dry powder weight 4.10 4.08 4.14 4.23 4.05 and standard deviation for each batch were calculated and The melting point of the compound proved too high to analysed using BP guidelines to assess batch quality. measure using the laboratory apparatus available (tempera- ture limit of 3508C). At 3308C, Ge-132 decomposed to a Disintegration tests brown solid with no visible droplets forming to indicate 22,57,58 melting. Disintegration testing was performed using a pre-assembled 6-well basket rack in a Copley Erweka ZT42 Disintegration 1 13 56 H and C NMR spectra of Ge-132 Apparatus maintained at 378C. Foreach batch, six randomly The H NMR (D O) spectrum showed two identical proton selected tablets were placed into the basket-rack and covered 2 with a 20 mm plastic disc. The basket was lowered into environments at 1.55 and 2.65 ppm, and the C NMR (D O) spectrum showed three distinct carbon environments 700 ml of immersion fluid and the apparatus operated for 2 at 178, 27 and 12.5 ppm. The peaks in both spectra were 15 min. The immersion fluids were 0.1 M HCl (pH 1.36) and deionized water (pH 7.4). The disintegration time was consistent with the proposed molecular structure and 57,58 monomeric formula of Ge-132. recorded as the time for the last of the six tablets to fully disintegrate. Disintegration tests were performed twice for Mass spectrum of Ge-132 each formulation in each of the immersion fluids. Hydrolysis of the compound resulted in numerous fragments Uniformity of content of varying mass presented in the full mass spectrum of Ge-132 (Fig. 2). The complex spectrum shows the relative Uniformity of content testing was carried out on abundances of distinct Ge-containing isotope clusters with Formulations 2–9 according to BP guidelines. Ten tablets splitting patterns very similar to those previously were individually weighed, crushed and dissolved in 59,60 reported. Additionally, these clusters occur in suggested 1000 ml of hot deionized water, then filtered to remove overlapping regions as indicated. These clusters indicate undissolved excipients. Two millilitres of samples were either incomplete fragmentation of the solid structure or analysed by UV–Vis (212 nm) and the absorbances used to formation of new linked Ge-132 units, possibly cyclic and calculate the content of Ge-132 in the tablets (against the 59–61 linear. standard calibration curve). The acceptance values (AVs) for deviations were calculated according to BP guidelines. FT-IR and FT-Raman spectroscopy of Ge-132 The FT-IR spectrum of the solid compound shows character- Results istic carboxylic O–H bond and C¼O bond vibrational peaks at 3300.00–2700.00 and 1687.55 cm , respectively Analysis of Ge-132 (Fig. 3). The peaks at 1410.00 and 1236.65 cm indicate Solubility, pH and melting point asymmetrical and symmetrical C–H bond vibrations of the Ge-132 fully dissolved in water forming an acidic solution of alkyl chains. Most notable in this mid-IR spectrum are the pH 3.06–3.12, and fully dissolved in common aqueous prominent characteristic sesquioxide peaks at 900.51 and laboratory solvents including 5% NaCl, 5% H SO and 800.04 cm , consistent with previous studies of pure 2 4 36,37 5% HCl. The compound was insoluble in common organic Ge-132 and its derivatives, confirming the –O–Ge– laboratory solvents (methanol, ethanol, diethyl ether and O–Ge-type network. chloroform) and only partially soluble in DMSO with The comparative FT-Raman spectrum of solid Ge-132 heating. (Fig. 4, top) shows more distinct carboxylic O–H bond ......................................................................................................................................................................................................................................... 132 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Figure 2. Full MS spectrum of pure Ge-132 showing suggested overlapping regions and % relative abundances of polymeric ions of Ge-132 in aqueous solution. Overlapping regions result from the polyisotopic nature of elemental Ge as well as loss or addition of neutral species (see text). A, monomeric ions; B, dimeric ions; C, region consisting of more complex trimeric, tetrametric, pentameric and hexameric ions. 21 21 Figure 3. FT-IR spectrum of Ge-132 solid showing % transmittance of O–H bonds (3300.00–2700.00 cm ) and C¼O bonds (1687.55 cm ) of the car- boxylic groups, –COOH. The peaks at 1410.00 and 1236.65 cm indicate C–H bond vibrations, and the characteristic sesquioxide peaks are visible at 900.51 and 800.04 cm . Figure 4. Comparative FT-Raman spectra of Ge-132 solid (top) and Ge-132 aqueous solution (bottom). Peaks in Ge-132 solid at 2932.90 (–COOH), 900.26 21 21 (sesquioxide) and 445.57 cm (–Ge–C–) are hydrolysed in solution to the broader 3361.96, 890.85 and 472.03 cm peaks, respectively. vibrations at 2932.90 cm as well as the characteristic aqueous Ge-132 in Fig. 4 (bottom) shows the vibrational sesquioxide peak at 900.26 cm . New peaks were frequencies of the hydrolysis products of Ge-132 with observed at 688.02 and 445.57 cm , the former peak indi- broader peaks observed at 3361.96, 1643.06 and 62 21 cating Ge–C bond vibrations. The Raman spectrum of 1437.44 cm . ......................................................................................................................................................................................................................................... 133 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... incorporated into the bulk powder had lower disintegration times (1 min 13 s to 1 min 36 s), though this was due to the smaller particle size. Uniformity of content test Mean absorbances at 212 nm ranged from 0.114 to 0.913. Using our calibration curve, we converted the absorbances into mean API content with the following results: Figure 5. UV–Vis calibration curve for Ge-132 at 212 nm for 0.00, 0.10, Formulation 2, 4.95%; Formulation 3, 4.68%; Formulation 0.20, 0.25, 0.50 and 1.0 mg/ml solutions. 4, 4.80%; Formulation 5, 5.02%; Formulation 6, 5.18%; Formulation 7, 2.45%; Formulation 8, 3.73%; and UV–Vis spectroscopy of Ge-132 Formulation 9, 0.65%. The mean % Ge-132 content for each formulation was then analysed using the BP formula for Between 215 and 210 nm, the serial dilutions we prepared calculating AV: showed consistent absorption within these wavelengths as well as relatively minimal variation in the gradient between jM  Xjþ ks the serial dilutions. For this reason, 212 nm was chosen as a suitable wavelength to study Ge-132 UV absorbance and where M is the reference value from the mean API content as a a resulting calibration curve produced (Fig. 5). Over 15 h, percentage of the label claim; X the mean content; k the a1mg/ml solution of Ge-132 had constant absorbance acceptability constant or tolerance interval (2.4 for 10 (A¼0.287) at 212 nm. tablets) and, s the standard deviation. An AV of 15 (termed L1) denotes a successful Analysis of tablet formulations formulation. Tablets of Formulations 1–5 measured 6 mm in diameter The AVs were calculated as 0.01, 4.87, 2.51, 0.00, 2.13, with a thickness of 2 mm, whereas Formulations 6–9 were 49.71, 24.49 and 86.07 for Formulations 2–9, respectively. slightly thicker at 4–5 mm with a similar surface diameter. Formulation 4 containing a 1:1 lactose:MCC mixture yielded a powder with better compressibility than Formulations 1–3 which had a 2:1 (lactose:MCC) mixture. Discussion This formulation was therefore chosen for further develop- Solubility, pH and melting (decomposition) point ment in Formulations 5–9. Ge-132, a white, crystalline powder, exists as a complex, infi- Uniformity of weight (Mass) test nite crystal network of O–Ge–O bonds with the basic Tablets of Formulations 2–9 passed BP test of Uniformity of monomeric formula O (GeCH CH COOH) (Fig. 6A). 3 2 2 2 Weight (Mass) with no sample deviating above or below Ge-132 fully dissolves in water and equilibrates to the trihy- the mean respective batch weight by .7.5%. Tablets in droxyl, germane triol, (OH) GeCH CH COOH (Fig. 6B), 3 2 2 Formulations 2–4 had mean weights of 116.45+ 3.06, forming an acidic solution (pH 3.06–3.12). Across all 110.33+ 4.06 and 109.38+ 3.75 mg, respectively. As tested solvents, water provided the best dissolution medium expected, tablets in Formulations 5–9 were heavier due to with the saturation solubility of Ge-132 determined to be incorporation of aqueous Eudragit . Mean weights were 1.28 g per 100 g of water. We also observed that even 123.40+ 3.15 mg (Formulation 5), 148.11+ 2.13 mg after cooling and over a period of 5 days, the compound (Formulation 6), 133.26+ 1.79 mg (Formulation 7), remained in solution and did not precipitate out. Stability 131.75+ 3.49 mg (Formulation 8) and 134.01+ 2.89 mg testing of the compound in solution over longer periods of (Formulation 9). time is an important aspect of future research which will provide further data on the viability of Ge-132 as a novel Tablet disintegration tests anticancer drug. Disintegration times for all formulations were determined in The melting (decomposition) temperature we measured both acidic and alkaline media. Tablets of Formulation 1 at 3308C is consistent with previously reported literature fully disintegrated within 13 s in both solutions which was and also expected from the known melting temperature expected, considering the powder did not contain significant of elemental Ge of 937.48C. The high melting amounts of binder. Formulations 2–4 had longer disinte- (decomposition) point is attributable to the crystal gration times, between 2 min 55 s in acidic pH and 3 min structure resulting from complex polymerization of the 10 s in alkaline pH. Formulations 5–9 with Eudragit monomeric unit. ......................................................................................................................................................................................................................................... 134 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Figure 6. (A) Structural representation of the basic monomeric unit of solid Ge-132, where R ¼ CH CH COOH, and n denotes the degree of polymerization 2 2 (adapted from Sawai et al. ). (B) Formation of germane triol (trihydroxylgermane) from hydrolysis of solid Ge-132. Figure 7. Proposed structures of some cyclic ions identified from peaks in the MS analysis of pure Ge-132 in aqueous solution, showing monomeric (A), dimeric (B and C) and trimeric (D and E) ion fragments of Ge-132 ( Ge) with predicted and actual m/z values. The loss of neutral species alters the shape of the ions, for example, structure C results from the loss of neutral CO and CH ¼CH from structure B (adapted from Wie et al. ). 2 2 2 NMR spectroscopy number of Ge atoms, and vary in mass depending on the 1 13 constituent Ge isotopes within each ion. H and C NMR were performed to confirm the structure of The smallest linear units are possibly the monomeric Ge-132. D O was chosen as the analysis solvent due to the þ þ ions (OH) Ge , (OH) GeCH CH and (OH) GeCH CH 3 3 2 2 3 2 2 favourable solubility in water. In the H NMR spectrum, COOH with calculated masses of m/z 125, 153 and 199, the triplet peaks at 1.55 and 2.65 ppm indicate proton respectively. These ions are present in our MS spectrum at pairs in the –Ge–CH –CH – and –CH –CH –COOH– 2 2 2 2 m/z 123.1, 151.1 and 193. Similarly, the basic cyclic units regions, respectively. These proton environments are dis- can be any of several different forms (Fig. 7A–E) ranging tinguishable by the proximity of the latter protons to the in mass from m/z 181 to 480. The peaks of these units are electronegative carboxyl oxygens, which shifts them further present in our spectrum within the monomeric, dimeric downfield on the chemical shift (d) scale. The d values are and trimeric regions from m/z 181 to 478.3. consistent with previously reported values of 1.61 and Another complication of interpreting the MS of Ge-132 is 2.69 ppm. fragmentation from addition or loss of neutral species—such The peaks presented in the C NMR spectrum were also as H O, CO ,CH ¼CH and GeO—can change the shape 2 2 2 2 consistent with the expected molecular structure and 57,58 and isomeric nature of the ion. For example, the prominent structural data for pure Ge-132 previously reported. peak at m/z 213.1 may be the linear dimeric fragment These peaks confirm the presence of carbons as follows: (OH) GeOGe , or the cyclic dimeric fragment in Fig. 7C 178 ppm, Ge–CH –CH –COOH; 27 ppm, Ge–CH – 2 2 2 resulting from loss of CO and CH ¼CH . Similarly, the 2 2 2 CH –COOH and, 12.5 ppm, Ge–CH –CH –COOH. 2 2 2 peak at m/z 193.0 could be the linear monomeric ion These d values are comparable to those previously as (OH) GeCH CH COOH , or possibly the linear dimeric 3 2 2 2 181.30, 28.43 and 15.68 ppm. ion HOGeOGeOH . The spectrum is further complicated by the polyisotopic nature of Ge where each isotope pro- MS spectrometry duces its own characteristic splitting pattern. This means Elemental Ge exists in five known stable isotopes— Ge that any combination of isotopes can be present in a frag- 72 73 74 (20.5%), Ge (27.4%), Ge (7.8%), Ge (36.5%) and ment causing overlapping peaks which may not be visible 76 61 Ge (7.8%). This polyisotopic nature produces a in the spectrum or identifiable in Fig. 2. complex MS spectrum with significant overlapping regions For the purposes of interpreting our spectrum, all masses consisting of different ions. Furthermore, these ions are poly- were calculated using the most abundant isotope, Ge, meric (monomeric, dimeric, trimeric, etc.) depending on the which accounts for the differences in m/z values between ......................................................................................................................................................................................................................................... 135 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... our calculated and measured masses. Further detailed MS Ge-132 tablet formulations analysis beyond the scope of this research is required to Preparation of most tablet formulations involves multistep accurately determine the exact isotopic composition of the processes including dry granulation, wet granulation and compound and conclusively ascertain the identity of the frag- fluid bed drying, prior to compression. However, due to ments. The spectrum however remains significant as it the small-scale nature of this study, preparation methods further confirms the purity and solubility products of our were limited to direct compression. All tablets were biconvex API and, most importantly, the absence of fragments of and produced by manual rotation of a single punch press. BP inorganic GeO . tests of resistance to crushing and tablet friability were not performed at the time due to technical failure of the available IR spectroscopy equipment. The higher loss in bulk powder weight prior to For additional confirmation of the purity and constituent compression in Formulations 5–9 can be attributed to the bonds in Ge-132, both FT-IR spectroscopy and FT-Raman extra processing steps (mixing, grinding, sieving and choos- spectroscopy were performed. Inorganic GeO absorbs ing a fraction of 75 mm) used to incorporate Eudragit strongest at 850 cm and produces a distinct characteristic into the bulk powder. peak in both FT-IR and Raman spectra. For identification Two interesting observations were made: first, despite pre- of functional groups which may not be visible in the FT-IR paring Formulation 5 with the reduced particle size and spectrum, FT-Raman spectroscopy was performed to detect increased die cavity depth, the tablets measured 6  2 mm, weaker vibrational frequencies in the far-IR region below similar to Formulations 1–4. It is possible that ES100 may 600 cm . Most notable is the peak observed at have altered the powder characteristics or the processing 445.57 cm ; however, without further analysis, the identity may have changed the flow and compaction properties of of this peak remains unknown and to date, there are no the powder, although the exact reason requires further inves- reported studies identifying this peak. Most important for tigation. Second, tablets of Formulation 6 were notably this research was conclusive confirmation of the absence of heavier (mean weight 148.11 mg) and thicker (5 mm), toxic, inorganic GeO (at 850 cm ). despite the same particle size and die cavity depth as The broader peaks at 3361.96, 1643.06 and Formulations 5, 7, 8 and 9. This may also be attributed to 1437.44 cm in the Raman spectrum of aqueous Ge-132 the constituent polymer (EL100) or the powder processing denote bond vibrations from –COOH and alkyl chain and emphasizes the need for further formulation studies. hydrolysis consistent with the MS fragments presented The fast disintegration times across all formulations are above. Of particular interest is the absence of the sesquioxide also attributable to the tablet manufacturing process using peaks at 800 and 900 cm previously seen in the FT-IR and dry granulation and direct manual compression of the dry Raman spectra of solid Ge-132, indicating hydrolysis of the powders. Wet granulation with a liquid binder to sufficiently crystal network and formation of newer –O–Ge–O– links wet the dry powders would be a more effective method of as seen in our MS. Finally, the peak between 890.86 and producing homogenous granules of API and excipients 708.49 cm in Fig. 4 (bottom) has been identified as charac- with better compaction properties. However, the disinte- teristic Ge–H bond vibrations within the trihydroxylger- gration times reported here provide a baseline for further for- mane, H–Ge–(OH) . The absence of most peaks mulation studies using alternative granulation methods. between 1400.00 and 601.37 cm is further evidence of Increasing the tablet dimensions by modifying the tabletting the formation of new hydrolysis products and, as seen machine parameters may also improve disintegration times previously, the GeO peak at 850 cm remains absent. as demonstrated by Obeidat et al. but should be made with consideration of the purpose of any oral formulation, UV–Vis spectroscopy bearing in mind various patient factors. Various wavelengths for UV–Vis spectroscopy (in water) There are various possibilities why Formulations 7–9 have been reported for Ge-132, from 190 to 210 nm. failed content uniformity testing, determined by the Because these wavelengths occur at the lower detection calculated AV. The polymer combinations within each for- limit of our spectrometer (190 nm), a series of scans from mulation may have altered the API characteristics; alterna- 220 to 190 nm were performed on dilute Ge-132 solutions. tively, the extra mixing step and decreased particle size At 212 nm, the absorbance series of the serial dilutions after polymer incorporation may have resulted in particle were most linear and this was chosen as a suitable wave- segregation which decreases powder quality and affects the length for this research. The 15 h (900 min) time-dependent distribution of API particles. Interestingly, a previous study UV–Vis absorbance at 212 nm showed constant absorbance using ES100–EL100 combinations as tablet coatings for (A ¼ 0.287), indicating no further decomposition of the the acidic drug mesalazine determined that a higher compound in solution and the overall stability of the EL100:ES100 ratio produced a more favourable drug-release hydrolysis products. profile when the ratio was kept above 4:1. ......................................................................................................................................................................................................................................... 136 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... Further large-scale studies using fluidized spray coating further large-scale formulation studies to assess the compat- will provide useful data on the compatibility of polymer ibility and suitability of polymers coatings such as Eudragit combinations with Ge-132 to formulate a novel gastro- with Ge-132, taking into account the acidic nature and solu- resistant anticancer treatment. The formulation studies bility characteristics of Ge-132. Additionally, further must also consider the reported solubility of Ge-132 in research should also appropriately determine Ge-132 solubi- water, the primary solvent used to prepare the polymer lity in simulated intestinal fluid or phosphate buffer (pH 6.8) suspensions. to mimic the natural passage of oral formulations through the GIT. Eudragit tablet formulations Finally, with advancements in nanotechnology and A range of Eudragit co-polymers are widely used to targeted drug delivery, future research might also focus on produce various modified-release profiles determined by the development of nano-formulations of Ge-132 beyond relative ratio of methacrylic acid to methyl methacrylate conventional gastro-resistant tablets. This research could ester within each polymer. In ES100, this ratio is 1:2 (acid:e- evaluate the compatibility of Ge-132 with various nanocar- ster), making it selectively soluble at pH .7.0 around the riers and formulation of the compound into novel nanopar- ileum and colon. The constituent ratio in EL100 is 1:1, ticulate systems such as Ge-132-polymer conjugates, making it soluble at a slightly lower pH (6–7) and useful liposomes and micelles. 52,53 for drugs targeting the jejunum. ES100 and EL100 have very similar physicochemical characteristics and for 51 Acknowledgements this reason were prepared in the same way. Dispersion of dry Eudragit powders in water and I would like to thank my project supervisors, Dr Katja addition of ammonia (1 M) produced a milky latex. This Stohfeldt-Venables and Dr Clare Rawlinson Malone, for consistency allowed us to gradually incorporate the their invaluable mentoring, guidance and supervision Eudragit dispersions dropwise into our dry powders as an during this exciting and challenging project. I would also alternative to wet granulation. In contrast, ES100 and like to acknowledge the Reading School of Pharmacy techni- EL100 formed glossy and highly viscous solutions in cal staff for the time they dedicated to providing support and ethanol which prevented dropwise addition. Moreover, use guidance throughout this study. of organic coatings is steadily declining in the pharma- ceutical industry due to environmental and health con- Funding cerns. Despite variable results from Formulations 5–9, future research of gastro-resistant Ge-132 formulations Funding and support for this project was provided by the should aim to maintain the particle size between 75 and School of Pharmacy, University of Reading, as part of the 180 mm, in addition to analysing the actual effect of Undergraduate Final Year Project module. polymers such as Eudragit on Ge-132. Author biography Conclusion and future studies S.M.O. graduated in July 2010 with an undergraduate The use of metals in medicine has significantly increased, Masters degree in Pharmacy from School of Pharmacy, particularly in developing novel anticancer therapies. The University of Reading, UK. She is currently undertaking anti-tumour activity of Ge-132 has been reported in numer- 1 year Pre-registration Pharmacist training in hospital phar- ous studies warranting further development as a novel macy. Special interests include paediatric pharmacy and anti-cancer drug. Despite the small scale of this study, we formulation of drugs for paediatric administration. were able to determine the purity of Ge-132 using Therapeutics areas of particular interest include cardiology, common spectroscopic techniques which are also efficient cancer and HIV. She is hoping to pursue a PhD in pharma- methods of detecting the presence of toxic GeO . ceutical formulation. Nearly, all anticancer drugs are administered parenterally, despite the oral route being the most popular route of admin- istration due to its ease, convenience and general patient References acceptability. Ge-132, a freely water-soluble compound, is 1. Ellis LM, Hicklin DJ (2009) Resistance to targeted therapies: refining antican- readily compatible with common tabletting excipients. cer therapy in the era of molecular oncology. Clin Cancer Res 15: 7471–7478. Although not a highly targeted formulation, the tablets 2. Minisini AM, Pauletto G, Andreetta C, Bergonzi P, Fasola G (2008) Anticancer presented here provide a convenient and simple novel oral drugs and central nervous system: clinical issues for patients and physicians. formulation for possible administration within a chemother- Cancer Lett 267: 1–9. apy regimen, supporting our main objective. Variable results 3. Vahid B, Marik PE (2008) Pulmonary complications of novel antineoplastic from tests of our gastro-resistant oral formulations warrant agents for solid tumors. 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Journal

Bioscience HorizonsOxford University Press

Published: Jun 6, 2011

Keywords: Ge-132 spectral analysis anticancer oral formulation

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