Development and validation of analytical methods for the determination of some drugs used for the treatment of colon diseases A thesis presented by Ahmed Adel Ibrahim Mohamed Othman B.Sc. Pharmacy and Biotechnology, Faculty of pharmacy, German University in Cairo 2011 Submitted for partial fulfillment for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry) Under the Supervision of Prof. Dr. Ramzia Ismail Ibrahim El-Bagary Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University Head of Pharmaceutical Chemistry Department, Faculty of Pharmacy-Future University Prof. Dr. Mohamed Mahmoud El-Kerdawy Professor of Medicinal Pharmaceutical Chemistry Faculty of Pharmacy, Mansoura University Dr. Ehab Farouk Hassan Elkady Assistant Professor of Pharmaceutical Chemistry Faculty of Pharmacy Cairo University Faculty of Pharmacy Cairo University 2017 English Abstract Development and validation of analytical methods for the determination of some drugs used for the treatment of colon diseases Different methods for the determination of some drugs used in the treatment of colon diseases, in bulk and dosage forms have been introduced in this thesis. The cited drugs are (balsalazide disodium, chlordiazepoxide, mebeverine hydrochloride, mesalazine, olsalazine sodium, and otilonium bromide). The chromatographic part includes a method for simultaneous determination of three commonly prescribed drugs used for treatment of inflammatory bowel diseases and was appled in ternary mixtures and dosage forms. Also, it involved simultaneous determination of olsalazine sodium and its degradation product 5-aminosalicylic acid (mesalazine) and was applied in binary mixtures and dosage form. Finally in this part, an HPLC method was introduced for the simultaneous determination of otilonium bromide and its degradation product 4- aminobenzioc acid and was applied in dosage form. On the other hand, spectrophotometric part includes simultaneous spectrophotometric determination of mebeverine hydrochloride and chlordiazepoxide in bulk and pharmaceutical dosage form using three chemometric techniques, classical least square(CLS), principal component regression (PCR) and partial least squares (PLS). Also, it involved simultaneous determination spectrophotometric determination of otilonium bromide with its degradation products 4- aminobenzoic acid and salicylic acid using CLS, PCR and PLS chemometric techniques. Also, this part includes derivative spectrophotometric methods in which a validated spectrophotometric method was introduced for simultaneous determination of mebeverine hydrochloride and chlordiazepoxide in binary mixture and pharmaceutical preparations using second derivative method for determination of mebeverine hydrochloride and first derivative method for determination of chlordiazepoxide and other validated fourth derivative spectrophotometric method used for simultaneous determination otilonium bromide with its degradation products 4-aminobenzioc acid and salicylic acid. Statistical analysis was carried out for comparing results of the proposed methods with the reference methods exhibiting good values and revealing insignificant difference. Keywords: balsalazide disodium, chlordiazepoxide, mebeverine hydrochloride, mesalazine, olsalazine sodium, otilonium bromide, HPLC, Chemometric techniques, derivative spectrophotometric methods, pharmaceutical preparations. I-1- General introduction Colon diseases include inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS) and colonic cancer. The chief types of inflammatory bowel disease are Crohn's disease (CD) and Ulcerative colitis (UC). Inflammatory bowel diseases fall into the class of autoimmune diseases, in which the body's own immune system attacks elements of the digestive system[1].The difference between CD and UC is the location and nature of the inflammatory changes. CD can affect any part of the gastrointestinal tract, from mouth to anus, although a majority of the cases start in the terminal ileum. UC, in contrast, is restricted to the colon and the rectum[2]. Microscopically, UC is restricted to the mucosa (epithelial lining of the gut), while CD affects the full thickness of the bowel wall ("transmural lesions"). Lastly, CD and UC present with extra-intestinal manifestations (such as liver problems, arthritis, skin manifestations and eye problems) in different proportions[2]. In spite of CD and UC being very different diseases, both may be present with any of the following symptoms: abdominal pain, vomiting, diarrhea, rectal bleeding, severe internal cramps/muscle spasms in the region of the pelvis and weight loss. Anemia is the most prevalent extraintestinal complication of inflammatory bowel disease [3, 4]. Associated complaints or diseases include arthritis, pyoderma gangrenosum, primary sclerosing cholangitis, and non-thyroidal illness syndrome [5]. Associations with deep vein thrombosis [6] and bronchiolitis obliterans organizing pneumonia have also been reported [7]. Diagnosis is generally by assessment of inflammatory markers in stool followed by colonoscopy with biopsy of pathological lesions. Colon associated diseases such as nervous colon or, IBS which is a group of symptoms including abdominal pain and changes in the pattern of bowel movements without any evidence of underlying damage [8]. These symptoms occur over a long time, often years [9]. There are three types of IBS: a- IBS with Constipation (IBS-C): this comes with stomach pain and discomfort, bloating, abnormally delayed or infrequent bowel movement, or lumpy/hard stool. b- IBS with Diarrhea (IBS-D): this comes with stomach pain and discomfort, an urgent need to move your bowels, abnormally frequent bowel movements, or loose/watery stool. c- IBS with alternating constipation and diarrhea [10]. Disorders such as anxiety, major depression, and chronic fatigue syndrome, are common among people with IBS [8, 11]. The causes of IBS are not clear. Theories include gut–brain axis problems, small intestinal bacterial overgrowth, genetic factors, food sensitivity, and gut motility problems [9]. Onset may be triggered by an intestinal infection [12], or stressful life event [13]. IBS is a functional gastrointestinal disorder [8]. Ulcerative colitis (UC) is a chronic inflammatory disorder of colon. This inflammation may be limited to the left-hand side of the colon or extend to entire colon. Colon related pathologies range in seriousness from constipation and diarrhea to the incapacitating inflammatory bowel diseases through to colon cancer, the third most widespread form of cancer in both women and men. Carcinoma of the colon afflicts patients with ulcerative colitis 7–30 times more frequently than it does the general population. The risk of colon cancer in ulcerative colitis is related to two factors: (1) duration of the colitis, and (2) extent of colonic involvement. The colonic malignancy associated with ulcerative colitis is generally an adenocarcinoma evenly scattered throughout the colon [14]. Figure (1): Anatomic distribution of Crohn’s disease and Ulcerative colitis [15] . 1-Some important drug classes of colon diseases Drugs used for treatment of IBS a-Muscarinic antagonists One of the most important compounds in this class is hyoscine, it acts on the relaxation of the smooth muscles of the gut [16], it is used as antispasmodic to reduce the activity of the gastrointestinal tract. Dicyclomine and hyoscyamine are also antispasmodics in IBS [16]. These medications can be given as an oral formulation or a sublingual tablet, and be dosed on an as-needed or regular basis. Many patients benefit by taking the medication before meals. A number of these agents are presented in Table (1). Mebeverine hydrochloride acts directly on the gut muscles at the cellular level to relax them [17]. This relieves painful muscle spasms of the gut without affecting its normal motility. Mebeverine possess a phenylethylamine moiety which affects the colonic motility as well as gastric motility and has been used in treatment of IBS [18]. Table (1). Table (1): Most commonly used Antispasmodics. Drug Structure Trade name Dicyclomine HCl .HCl Spasmorest® Hyoscine N- Buscopan® Butylbromide Mebeverine Coloverin SR® Otilonium bromide Spasmomen® b-Calcium antagonists Pinaverium Bromide belongs to this class which inhibits the calcium influx by blocking the voltage dependent calcium channels at the smooth muscle cell level. It is selective for the intestinal smooth muscle [19]. It is used to treat IBS by relieving the symptoms of abdominal pain, bowel disturbances and intestinal discomfort [20]. Structure activity relationship (SAR) study reveals that quaternary ammonium derivatives such as pinaverium bromide have been discovered and developed as potent spasmolytic of the gastrointestinal tract. Table (2). Table (2): Most commonly used Calcium antagonists. Drug Structure Trade name Pinaverium Bromide Spascolon® c-Adjuvant therapy for IBS Although many drugs are available to treat the symptoms of irritable bowel syndrome, these drugs do not cure the condition. They are primarily used to relieve symptoms. The choice among these medications depends in part upon whether the patients have diarrhea, anxiety or pain-predominant irritable bowel syndrome such as: 1-Anti-anxiety drugs Anti-anxiety drugs reduce anxiety. Chlordiazepoxide (Librax®), Diazepam (Valium®), lorazepam (Ativan®), and clonazepam (Klonopin®) belong to this class of drugs. Anti-anxiety drugs are occasionally prescribed for people with short-term