Trace amounts of pharmaceuticals have been detected in water, from nanograms per liter to micrograms per liter, and have a negatively effect in the aquatic environment and an increased potential risk of drug poisoning for human and animals. In order to address the problem, drug degradation catalyzed by chloroperoxidase (CPO) has been investigated. CPO is a heme-containing glycoprotein secreted by the fungus, Caldariomyces fumago, it catalyzes two major types of oxidations, two one-electron oxidations as catalyzed by most peroxidases and two-electron oxidations which are rare for conventional peroxidases. Five common drugs from a variety of classes which were persistent in the environment have been studied. The metabolites of each drug were identified and the pathways of degradation were proposed. All of them were found to be 100% degradation efficiency in the CPO-H2O2-Cl- system which the catalyzation only required low concentration of CPO (normally nanomolar level) as well as relatively low concentration of H2O2 as cofactor. This degradation method is economic and highly efficient, the results of my experiment extensively support the hypothesis that CPO has a great potential in the environmental application. A new mutant of CPO has been constructed to investigate the role of histidine 105 in the active site of distal pocket. Histidine 105 was suggested to play an essential role in modulating the chlorination activity by forming hydrogen bond with glutamic acid 183, histidine has been replaced by arginine to generate CPO H105R mutant. The construction and transformation were a success but the protein was expressed as apoenzyme, suggesting the mutagenesis to a larger arginine residue at position105 disturbed the heme incorporation.

A number of cyclic peptides containing a positively charged ring composed of arginine residues attached to hydrophobic tail made of tryptophan residues through a lysine linker namely [R5K]W5, [R6K]W5, [R5K]W6, [R7K]W5, [R5K]W7, [R6K]W6, and [R7K]W7 were synthesized and evaluated as molecular transporters. The peptides were evaluated for their ability to deliver, fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI), and fluorescent labeled anti-HIV drugs (F′-FTC and F′-d4T). The results indicated that the presence of positively charged arginine residues on the ring and hydrophobic tryptophan residues in a sequential linear outside the ring was an optimal approach to improve the intracellular uptake of cargo molecules through non-covalent interactions. Some of these peptides were also evaluated for their efficiency for intracellular delivery of siRNA to triple-negative breast cancer cell lines in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). [R6K]W6 and [R5K]W5 were found to be very efficient in the delivery of siRNA. Furthermore, co-formulation of peptides with lipid DOPE significantly enhanced the efficiency of siRNA delivery compared to peptide alone. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. A chemotherapeutic drug, doxorubicin (Dox) was covalently conjugated to the cyclic peptide [R5K]W7A and linear peptide R5KW7A, and the biological activity was evaluated in cell-based assays. Comparative antiproliferative assays between covalently conjugated peptide-Dox and the corresponding noncovalent physical mixtures of the peptides and Dox were performed. The conjugation of Dox with cyclic [R5K]W7A-Dox exhibited similar antiproliferative activity compared to Dox alone after 72 h incubation time in all cancer cell lines, such as leukemia, ovarian and gastric cancer cells. However, [R5K]W7A-Dox significantly reduced the cell cytotoxicity in normal cell lines such as normal heart muscle and normal kidney cells after 72 h when compared with Dox alone. These results revealed that this cyclic peptide prodrug can be used as a potential candidate for the treatment of cancer cells with reduced side effects against normal cells in the body.

<p>Therapeutic proteins are often lyophilized with excipients such as sucrose or trehalose to protect them during manufacturing and achieve a longer shelf-life. Formulation design for therapeutic proteins has been a trial-and-error process, and the mechanisms responsible for the stabilizing effects of excipients are not fully understood. Two proposed theories have been widely accepted: the water replacement theory and the vitrification theory.^1,2The water replacement theory suggests that excipients stabilize protein molecules in the solid state by forming hydrogen bonds that “replace” the hydrogen bonds to water that stabilize the protein in solution, while the vitrification theory asserts that proteins are stabilized by a glassy solid matrix of low mobility and does not require direct interactions between excipient and protein. A better understanding of the interactions between proteins and other components of the lyophilized matrix can facilitate rational formulation design and shorten the time in development. However, most of the analytical methods available can only provide information on the bulk properties of the lyophilized matrix such as moisture content and glass transition temperature (<i>T</i>_g); it has been difficult to measure the interactions between protein and excipient directly, if they exist. In order to characterize the interactions between protein and excipients in a lyophilized matrix with high resolution, a photolytic labeling method was developed in this dissertation, building on previous work in our research group. Photolytic labeling has long been used to identify protein-protein interactions <i>in vivo</i>.^3,4Common types of photo-reaction reagents and their applications are summarized in Chapter 1. The research described in this dissertation utilizes the diazirine functional group, which is activated after UV exposure and undergoes a free radical reaction to form covalent bonds with nearby molecules. The reaction can be used to identify the interactions between excipients and protein or peptide in a solid formulation. Previous studies in our lab have shown that photo-reaction can be applied to lyophilized solids to study protein-matrix properties and interactions in the solid.^5,6This dissertation seeks to further identify photo-reaction products and analyze them in a more quantitative way. </p><p> </p><p>Chapter 2 describes a quantitative analysis of photo-reaction products in solution and lyophilized solids using a model peptide, KLQ (Ac-QELHKLQ-NHCH₃). The purpose of the work in this chapter is to establish a quantitative analytical method for photo-reaction products, enabling studies of peptide-excipient interactions in lyophilized solids. KLQ was derivatized with a bifunctional probe NHS-diazirine (succinimidyl 4,4’-azipentanoate; SDA) at Lys5 to be photo-reactive. The SDA derivatized KLQ (KLQ-SDA) was used to study the photo-reaction products and examine excipient interactions. Identification and quantitation of photo-reaction products of KLQ-SDA was achieved with liquid chromatography mass spectrometry (LC-MS) and reversed phase HPLC (rp-HPLC). Important reaction products such as peptide-excipient adducts and peptide water adducts varied in different formulations. Unexpected reaction products such as unproductive “dead-end” products and peptide-phosphate adducts from buffer salt were also detected and quantified. Together, the photo-reaction products reflected the local environment near Lys5 of the peptide in the solid state. This study has provided a better understanding of photo-reaction with diazirine in the lyophilized solids together with a quantitative description of the local environment near Lys5. </p><p> </p><p>In Chapter 3, the photo-reaction products in lyophilized solids exposed to increasing moisture were analyzed, and the effect of increasing moisture on the local environment near the peptide was examined. Using the analytical method developed in Chapter 2, these studies explored whether peptide-water interactions, as measured by the formation of water adducts formed by photolytic labeling, are linearly correlated with an increase in solid bulk moisture content. Formulations containing the KLQ-SDA peptide were exposed to various relative humidity conditions and photolytic labeling was induced. Solids containing disaccharide excipients behaved differently from those containing amino acids when exposed to the same relative humidity condition, showing different levels of peptide-excipient and peptide-water adducts. With increasing moisture content in the solids, the formation of photo-reaction products did not mimic the pattern of solutions with same composition, indicating differences in the local environment. </p><p> </p><p>An alternative approach to studying lyophilized formulations using photolytic labeling is to incorporate photo-reactive excipients into the solid matrix. In Chapter 4, a new diazirine-labeled photo-excipient, photo-glucosamine (pGlcN), was chemically synthesized and incorporated into formulations of the therapeutic peptide salmon calcitonin (sCT) and compared with the commercially available diazirine-labeled amino acid, photo-leucine (pLeu). The studies in Chapter 4 further compared peptide-excipient interactions at the molecular level with two different photo-excipients, ionizable pLeu and unionizable pGlcN. Changing solution pH prior to lyophilization was expected to change ionic interactions between sCT and pLeu in the solid samples, resulting in different distributions of photo-reactions products; pH-dependent differences were not expected for pGlcN. The results demonstrated that the distribution of photo-reaction products varied with the composition of the formulation and the pH of the solution prior to lyophilization. The photo-reaction products in the pGlcN-containing formulation differed from those pLeu, showing a difference in the interactions of unionizable (pGlcN) and ionizable (pLeu) excipients with sCT in solid samples. </p><p> </p><p>The work in this dissertation has developed photolytic labeling as a tool to study lyophilized peptide formulations, and has provided a more quantitative understanding of the photo-reaction products that are produced from diazirine-labeled peptides or excipients in the solid state. A new photo-reactive excipient has also been presented (pGlcN), which showed different photo-reaction products than a commercially available photo-excipient (pLeu) and is promising for future study. Photolytic labeling for formulation development is still in its early stages, and additional research regarding reaction mechanism and complementary stability studies is needed. Nevertheless, the results presented in this dissertation support continued development of photolytic labeling as a practical method for formulation design and development. </p><p> </p>

Apurinic/apyrimidinic DNA repair endonuclease-1 (APE1), first recognized as an important DNA excision repair enzyme, is also known as Redox Factor-1 (Ref-1) involved in the activation of many nuclear transcription factors in both redox-dependent and independent manner. It has been well-documented that the overexpression of APE/Ref-1 contributes to the development of chemo-resistance and is associated with tumor progression in many human malignancies [1]. Our previous study in melanoma demonstrated that the development of novel inhibitors targeting the redox regulation domain of APE/Ref-1 is a promising strategy for melanoma treatment. To date, limited successes have been reported in developing novel APE/Ref-1 inhibitors for cancer treatment. Utilizing a structure-based approach, our study identified and characterized small molecular inhibitors of APE/Ref-1. First, N-terminally truncated APE/Ref-1 protein lacking the first 40 amino acid residues (∆40APE-1wt) was cloned into the pGEX-6P1 vector to express the GST-∆40APE-1wtprotein. After cleavage of GST-tag, the concentrated ∆40APE-1wt protein was subjected to protein crystallization study. We have successfully diffracted ∆40APE-1wt crystals and collected data with a resolution of 1.57Å. The crystal structure was further determined by molecular replacement in Molrep using the already available human APE-1 structure (PDB: 5CFG). For the first time, we observed the dimerization of APE/Ref-1 protein formed under oxidative conditions, which may contribute to the redox regulation of APE/Ref-1. Such structural transformation of APE/Ref-1 protein under distinct redox conditions may pave the way for future drug development and optimization. The binding affinity of the candidate compounds with ∆40APE-1wt protein was also determined using Surface Plasmon Resonance (SPR), and the Ki values were analyzed. One of the potent inhibitors developed by our group by structure-based approach, exhibited promising anti-melanoma activities both in vitro and in vivo. Future studies on the structure-activity association are warranted.

Introduction: Genetic rearrangements in Ewing sarcoma, prostate, and leukemia cells result in activation of oncogenic ETS transcription factor fusions. Mithramycin (MTM) has been identified as an inhibitor of EWS-FLI1 transcription factor, a gene fusion product responsible for oncogenesis in Ewing sarcoma. Despite preclinical success, a phase I/II clinical trial testing MTM therapy in refractory Ewing sarcoma was terminated. Liver and blood toxicities resulted in dose de-escalation and sub-therapeutic exposures. However, the promise of selectively targeting oncogenic ETS transcription factors like EWS-FLI1 prompted us to undertake the discovery of more selective, less toxic analogues of MTM. MTM is a potent inhibitor of ubiquitous SP1 transcription factor, likely inducing non-specific toxicity. In collaboration with two medicinal chemistry groups, two semi-synthetic efforts were implemented to develop novel analogues of MTM. The first effort utilized the biosynthetic product mithramycin SA (MTMSA) to modify C3-side chain. The second effort utilized an oxime linker directly formed on MTM’s C3-side chain (MTM-oxime; MTMox). Here I present the pharmacological assessment of over 75 novel MTM analogues towards selectively targeting oncogenic ETS transcription factors, like EWS-FLI1, over ubiquitous transcription factors, like SP1. Methods: Novel MTM analogues were evaluated for selective cytotoxicity against ETS fusion-dependent cell lines. Selectively cytotoxic analogues were evaluated for inhibitory effects on several gene promoters in TC-32 reporter cell lines, a Ewing sarcoma cell line dependent on EWS-FLI1, transfected with luciferase reporter vector. Cloned reporter vectors incorporated NR0B1 (EWS-FLI1 binding), β-actin (SP1 binding) and CMV (non-specific) gene promoters. Furthermore, gene (mRNA) and protein expression changes of EWS-FLI1 and SP1, as well as regulated target genes, namely NR0B1, VEGFA and BCL-2 were evaluated with MTM analogue treatments. The MTM analogues with most selective activity in vitro were administered to mice by intravenous bolus dose for pharmacokinetic analysis. The MTM analogues with highest systemic exposure from each semi-synthetic effort, namely MTMSA-Trp-A10 and MTMox-24, were further evaluated. Metabolic stabilities in whole blood, plasma, and tumor cell matrices, and across multiple species were compared with MTM. Moreover, intrinsic hepatic clearances were estimated using mouse liver microsomes. Tumor and liver distributions were estimated in tumor bearing mice. Additionally, the effect of organic anionic transporter polypeptides (OATP) on distribution of MTM was investigated. Maximum tolerated doses were evaluated for lead MTM analogues, having both selective activities in vitro and high systemic exposure, compared to MTM. Complete blood cell counts and plasma alanine aminotransferase activity were measured to evaluate dose-dependent blood and liver toxicities, respectively. ETS fusion-dependent and non-dependent cell lines were implanted subcutaneously into immunocompromised mice for efficacy studies. Average tumor volumes and survival were tracked for mice receiving treatment, compared to MTM and vehicle treatment. Results: Evaluation of MTM analogues from both semi-synthetic efforts revealed that conjugation of MTM C3-side chain with tryptophan (Trp) and/or phenylalanine (Phe) improved selective cytotoxicity against ETS fusion-dependent cell lines. This was highlighted by MTMSA-Trp-A2 (also refer to as MTMSA-Phe-Trp) and MTMSA-Trp-A10 (also refer to as MTMSA-Trp-Trp), with selective indices of 19.1 and 15.6, respectively, compared to MTM (1.5). Similarly, MTMox-23 (also refer to as MTMox-Phe-Trp) and MTMox-20 (also refer to as MTMox-Trp) had selectivity indices of 4.6 and 4.5, respectively. These selectively cytotoxic MTM analogues inhibited EWS-FLI1-mediated transcription 10-fold more effectively than both non-specific CMV-mediated and SP1-mediated (via β-actin promoter) transcription in TC-32 reporter cell lines. Moreover, gene (mRNA) and protein expression of EWS-FLI1 and regulated gene, NR0B1, were inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Similarly, SP1 and target genes, VEGFA and BCL-2, gene (mRNA) and protein expressions were also inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Conjugation of Trp and/or Phe to C3-side chain of MTM increased systemic exposure in vivo. Most impressively, the addition of two Trp residues, namely MTMSA-Trp-A10 and MTMox-24 (also refer to as MTMox-Trp-Trp), resulted in systemic exposure increases of 218- and 42-fold, respectively, after intravenous (IV) bolus dose. Metabolically, tryptophan/phenylalanine conjugated MTM analogues are liable to esterase activity on carboxy-methyl functional group. Very rapid de-methylation in biological matrix was observed with MTMox-24, compared to MTMSA-Trp-A10, suggesting a regiospecific effect. However, esterase activity was limited to rodent matrices and demethylation occurred at significantly diminished rates in non-human primate and human plasma. MTM analogues were not susceptible to p450-mediated metabolism, with negligible loss in mouse liver microsome assay compared to verapamil control. MTM (1mg/kg) and MTMox-24 (6mg/kg) were detected in subcutaneously implanted (flank) LL2 tumors and liver homogenates after IV bolus dose. Interestingly, MTMSA-Trp-A10 (2mg/kg) was not. Despite a 3-fold increase in systemic exposure with rifampin oral pretreatment, an OATP inhibitor, exposure of MTM was unaffected in Oatp knockout mouse model. Exposure of MTM in liver tissue was 8.4-fold higher compared to tumor tissue with low tissue clearance. This agrees with the lack of metabolism observed in liver microsomes and may provide a mechanism for clinically observed liver toxicity. MTMSATrp-A10 had a single maximum tolerated dose (MTD) of 0.75mg/kg, compared to 1mg/kg for MTM, administered by IV bolus. In contrast, MTM-oxime analogues (MTMox-20, -23, -24 and -25) had single maximum tolerated doses of 20 – 25mg/kg. These increased tolerances are the result of additive differences in whole blood stability, cytotoxicity and systemic exposure. At a dose of 0.75mg/kg, administered every 3 days, MTMSA-Trp-A10 did not result in an efficacious result in tumor xenograft studies. These studies remain under further investigation, but the result may indicate high plasma protein binding of MTMSA-Trp-A10 and lack of free fraction available within tumor. The most selective MTM-oxime analogue in vitro, MTMox-23, significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0025, day 16, one-way ANOVA multiple comparisons test) compared to MTM (p=0.1174, day 16) and extending survival for 17 days out of 48 days on study (p=0.0003, Log Rank (Mantel-Cox) single comparison test) with treatment at MTD every 3 days, compared to vehicle. Additionally, the MTM-oxime analogue with highest systemic exposure, MTMox-24, also significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0003, day 21, one-way ANOVA multiple comparisons test) compared to MTM (p=0.032, day 21) and extending survival for 12 days out of 37 days on study (p=0.0004, Log Rank (Mantel-Cox) single comparison test) with treatment, compared to vehicle. Conclusion: These studies in whole highlight the importance of exposure (pharmacokinetics; PK), toxicity and efficacy (pharmacodynamics; PD) relationships. The cytotoxicity and high systemic exposure of MTMSA-Trp-A10 directly contributes to its lower tolerated dose. However, despite a similar tolerated dose to MTM, systemic exposure remains 163-fold higher at the MTD. High systemic exposure may be attributed to high plasma protein binding, but also reduces the exposure of free MTMSA-Trp-A10 within the tumor tissue, which drives the efficacious response. In contrast, the less cytotoxic and rapidly de-methylated MTM-oxime analogues allow for 25-fold higher tolerances in mice. This unique metabolism and clearance may prevent exposures required to induced systemic blood and liver toxicities induced by MTM. Moreover, at these highly tolerated doses, the initial systemic exposure at MTD is highest among analogues tested, which resulted in an efficacious response with MTMox-23 and MTMox-24 treatment in tumor xenograft models. It remains to be determined if these PK/PD relationships can be reproduced in additional animal models, including human, without inducing toxicity. Nonetheless, these initial studies in mice demonstrate that a more selective, more tolerated analogue of MTM has potential for clinical success in treating ETS fusion-dependent tumors.

No description available.

<p>Sialic acid synthase (NeuB) is a key enzyme in bacterial biosynthesis of the sialic acid <em>N</em>-acetylneuraminic acid (NeuNAc). It catalyzes the addition of phosphoenolpyruvate (PEP) to <em>N</em>-acetylmannosamine (ManNAc) in the presence of a divalent cation such as Mn²⁺. We have explored the inhibition of NeuB by an oxacarbenium ion mimic, NeuNAc oxime, and hydroxylamine (NH₂OH). NeuNAc oxime shows slow-binding inhibition with a binding half-life of 2.5 h and an inhibition constant (<em>K</em>_i^*) of 1.6(± 0.7) pM. Even though NeuNAc oxime binds NeuB with high affinity, there remains approximately 10% residual activity even after extended pre-incubation with high inhibitor concentrations. In contrast, in the presence of substrates, when NeuB was actively catalyzing NeuNAc synthesis, complete inhibition by NeuNAc oxime was observed within 6 h. This inhibition profile is similar to NH₂OH; which has previously been shown to elicit complete, time-dependent inhibition. We propose the existence of two NeuB conformations: an asymmetric idle state conformation (NeuB^IS), in which NeuNAc oxime is able to bind to only one monomer of this dimeric enzyme, and a second conformation, running state NeuB (NeuB^RS), which is completely inhibited due to either NeuNAc oxime binding to the second monomer, or the dimer adopting a conformation in which the unbound monomer is inactive. Experiments with [1-¹⁴C]PEP showed that in the presence of large excess of substrate, inhibition occurred faster than with a lower excess. This suggests that a sustained buildup of NeuB^{RS<strong> </strong>}is required for complete inhibition.</p> / Master of Science (MSc)

<p>Nonsteroidal anti-inflammatory drugs are among the most commonly used prescription and over-the-counter medications, but they often produce significant gastrointestinal ulceration and bleeding, particularly in elderly patients and patients with certain co-morbidities. Novel anti-inflammatory drugs are seldom tested in animal models that mimic the high-risk human users, leading to an underestimate of the true toxicity of these drugs. In the present study we examined the effects of two novel NSAIDs and two commonly used NSAIDs in models in which mucosal defence was expected to be impaired. Naproxen, celecoxib, ATB-346 (a hydrogen sulfide- and naproxen-releasing compound) and NCX 429 (a nitric oxide- and naproxen-releasing compound) were evaluated in healthy, arthritic, obese, hypertensive rats, and in rats of advanced age (19 months) and rats co-administered low-dose aspirin and/or omeprazole. In all models except hypertension, greater gastric and/or intestinal damage was observed when naproxen was administered in these models than in healthy rats. Celecoxib-induced damage was significantly increased when co-administered with low-dose aspirin and/or omeprazole. In contrast, ATB-346 and NCX 429, when tested at doses that were as effective as naproxen and celecoxib in reducing inflammation and inhibiting cyclooxygenase activity, did not produce significant gastric or intestinal damage in any of the models. These results demonstrate that animal models of human co-morbidities display the same increased susceptibility to NSAID-induced gastrointestinal damage as observed in humans. Moreover, two novel NSAIDs that release mediators of mucosal defence (hydrogen sulfide and nitric oxide) do not induce significant gastrointestinal damage in these models of impaired mucosal defence.</p> / Master of Science (MSc)

<p>Colorectal cancer leads to more than 600,000 deaths worldwide per year. An abundance of research has shown that several non-steroidal anti-inflammatory drugs (NSAIDs) can exert chemotherapeutic and chemo-preventative effects in colorectal cancer patients. It is important to note, that use of many different NSAIDs carries a significant risk for cardiovascular and gastrointestinal (GI) complications. A recently developed group of NSAIDs, which release hydrogen sulfide (H₂S), has been shown to have greatly reduced these side effects as compared to conventional NSAIDs. This is likely attributable to the ability of H₂S to increase the resistance of the GI mucosa to injury, as well as to accelerate repair of injury when it occurs. Moreover, H₂S has been shown to be a vasodilator, and therefore may offset some of the hypertensive effects of NSAIDs.</p> <p>We assessed the chemotherapeutic actions of two of these newly developed NSAIDs. ATB-346 is an H₂S-releasing derivative of naproxen and ATB-352 is an H₂S-releasing derivative of ketoprofen. These drugs were tested in the azoxymethane mouse model and in the APC^Min/+mouse model of Colorectal cancer.</p> <p>In the azoxymethane model of colorectal cancer ATB-346 caused a significant reduction in number aberrant crypt foci (ACF), which are pre-neoplastic lesions used as markers of colorectal cancer. The reduction was superior to naproxen at all doses tested. ATB-352 also caused a significant reduction in the number of ACF, however the reduction was not superior to that produced by ketoprofen. In APC^Min/+mice treated with ATB-346 for 14 days (14.5 mg/kg) we observed a complete inhibition of the formation of colonic polyps/tumours and a 97.5% reduction in total polyp score. Shorter treatment with ATB-346 also produced similar reduction in total polyp score. We found that ATB-346-treated mice had lower levels of b-catenin and cmyc without significant changes in APC or p53 levels. <strong></strong></p> <p>These results demonstrate ATB-346 can exert superior chemo-preventative effects in mice models of colon cancer while leading to no gastric or intestinal damage.</p> / Master of Science (MSc)

High shear melt granulation (HSMG) is an established technology for a production of densified granules. In this project, it was used as a novel solvent-free method for the preparation of cocrystals. Cocrystals produced by HSMG were compared to those prepared by Hot Melt Extrusion (HME) to investigate the influence of variable parameters and conditions on the process of cocrystal conversion. The potential for the active control of cocrystals polymorphism utilising the intrinsic properties of lipids was also investigated in this project. Different cocrystal pairs were prepared by both cocrystallisation methods using glycol derivative polymers. Thermal analysis, powder X-ray diffraction and Raman spectroscopy were used as analytical techniques to determine the cocrystal yield and purity. The results obtained from HSMG suggest that sufficient binder concentrations (above 12.5% w/w) in a molten state and continuous shearing force are necessary to achieve a complete cocrystals conversion. Further increase in binder concentration (15% w/w) was found to provide more regular shape and smooth surface to the prepared spherical granules. Cocrystals preparation by HME was achievable after introducing a mixing zone to the extruder configuration (Conf B and Conf C) providing densified extrudates containing pure cocrystals. In conclusion, HSMG was found as a versatile technique for the preparation of pure pharmaceutical cocrystals embedded in polymer matrix within a spherical shape granule of smooth surfaces, providing additional desirable characteristics. Intensive surface interaction, enhanced by sufficient mixing under optimal parameters, was found as a key influencing factor in cocrystallisation. Cocrystals polymorphism was actively controlled by employing the intrinsic properties of polymers and lipids.