THE DISTRIBUTION OF IPRONIAZID IN BODY FLUIDS AND TISSUES AFTER ORAL ADMINISTRATION AND A METHOD FOR ITS DETERMINATION DISSERTATION Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By CARL EDWARD MOYER, B. S., M. S. <1 The Ohio State University 1959 Approved by Department of Physiological Chemistry ACISOWLEDGSKEHT The author wishes to express his sincere appreciation to Dr. Helen L. Wikoff for her counsel, interest and patience in preparing this work. ii TABLE OP CONTENTS Page Introduction............................................ 1 Historical.............................................. 4 Preparation and Chemical Properties of Iproniazid.... 8 General Methods for the Determination of Carboxylic Acid Derivatives........................... 11 Experimental Procedures and Results................. 14 A. Attempts to Develop Colorimetric Procedures for the Determination of Iproniazid................................... 14 B. Spectrophotometric Procedures............. 16 C. Attempts to Separate Iproniazid from Pyridine Carboxylic Acids and Their Derivatives............................... 19 D. The Determination of Iproniazid in Aqueous Solutions........................................ 2 4 E. Quantitative Estimation of Iproniazid Added to Pooled Serum................ 26 P. Quantitative Estimation of Iproniazid Added to Urine.................................. 32 G. Procedure Finally Adopted for the Quanti­ tative Determination of Iproniazid In Blood and Urine................................. 39 1. Reagents................................ 39 2. Procedure............................... 40 iii iv Page H. The teOiantitative Estimation of Iproniazid In Rabbit Serum Following Oral Admini­ stration......................................... lj.2 I. Determination cff the Amount of Iproniazid Excreted in the Urine............... i+9 J. Determination of Iproniazid In Serum And 21; Hour Urine Specimens From Psychotic Patients.............. 53 K. Absorption of Iproniazid From the Gastro- intestional Tract of a Rat.................... 55 L. Determination of Iproniazid In Body Tissues.................................... 57 Discussion.............................................. 59 Summary................................................. 61; Bibliography............................................ 67 Autobiography........................................... 70 LIST OP TABLES Table Page 1 The Amounts of Iproniazid, Isonicotinic Acid anc: Isonicotinic Acid Amide Extracted by Organic Solvents from 1 ml. Portions of Aqueous Solutions Containing 25 Gamma of the Respective Compounds.......................... 20 2 The Amount of Iproniazid Extracted Prom 1 ml. of An Aqueous Solution Containing 25 Gamma Iproniazid by 50 ml. Chloroform After the Addition of 1 ml. of Various Bases in the Concentrations Listed.............................. 21 3 Effect of Time of Extraction With Chloroform on Iproniazid Recovery Prom 1 ml, Basic Aqueous Solution Containing 25 Gamma Iproniazid Per ml.. 22 Lj. Amounts of Iproniazid Recovered Prom Basic Aqueous Solutions by Various Quantities of Chloroform.......................................... 22 5 Effect of Concentration of Hydrochloric Acid on the Amount of Iproniazid Recovered Prom 25 ml. Chloroform Solution Containing 22 Gamma Iproniazid Per ml .................................. 23 6 Time Necessary to Extract Iproniazid Prom 25 ml. Chloroform'With 5 ml. of IN Hydrochloric Acid................................................ 2l| 7 The Determination of Iproniazid in Aqueous Solutions........................................... 26 8 The Amount of Added Iproniazid Pound in 1 ml. Solution When the Iproniazid Standard Was Extracted in the Same Manner...................... 26 9 Absorbance at 268 nyt Shown by 1 ml. Samples of Sera Obtained Prom Ten Normal Persons.............. 27 v vi Table Pag© 10 Amount of Iproniazid Recovered From 1 ml. Serum Samples Containing 25 Gramma Iproniazid... 28 11 Effect of Time of Extraction With Chloroform On Iproniazid Recovery From 1 ml. Serum Containing 25 Gamma Per ml ....................... 28 12 Stabilization of the Recovery of Iproniazid From Serum by the Addition of Ammonium Salts... 29 13 Effect of the Addition of Various Quantities of Ammonium Chloride on the Chloroform Extraction of luroniazid From 1 ml. Serum Containing 25 Gamma per ml ......................... 30 lit Determination of Iproniazid In 1 ml. Pooled Serum............................................... 30 15. The Amount of Added Iproniazid Found in 1 ml. Serum When Compared With a Standard Dissolved in Serum............................................ 32 16 Minimum Amount Iproniazid That Can Be Determined Accurately In 1 ml. Serum........... 32 17 Absorbance at 268 my Produced by Extracts From Normal Urine Treated According to the Procedure for the Isolation of Iproniazid 33 18 Effects of Various Concentrations of Ammonium Hydroxide Added to Urine Before Extraction With Chloroform.................................. 33 19 Effect of Various Amounts of Ammonium Chloride Added to Urine Before Extraction With Chloroform on the Absorbance at 268 irjy.... 3^ 20 Absorbance at 268 nyu Produced by 5 ml. Aliquots of 2l± Hour Urine From 57 Individuals........... 35> 21 Comparison of the Amounts of Iproniazid Found in 5 ml. Urine With That Added.................. 37 22 The Amount of Added Iproniazid Found in 5 ml. Urine............................................. 37 vli Table Page 23 The Amount of Added Iproniazid Pound in 5 ml. Urine Specimens When the Standard Consisted of Iproniazid Dissolved in Urine.................. 38 2[|_ Absorbance at 268 mjf Produced by Extracts Prepared Prom the Serum of a Rabbit According to the Procedure For the Isolation of Iproniazid....................................... J4J4- 25 Absorbance and Amount of Iproniazid in the Serum of a Rabbit 2 Hours After Receiving 1.6 mg. Iproniazid............................... 26 Iproniazid Content of Rabbit Serum Drawn at Various Intervals After Oral Administration of Iproniazid.................................... lj.6 27 Concentration of Iproniazid in Rabbit Serum Following the Daily Administration of 2.0 mg. Iproniazid Phosphate Per kg. Body Weight..... 1+8 28 Concentration of Iproniazid in Rabbit Serum Following the Daily Administration of 1|,.0 mg. Iproniazid Phosphate Per kg. Body Weight 1+9 29 Amounts of Iproniazid Found in 21+ Hour Urine Specimens Obtained From Two Rabbits That Had Received Daily Oral Doses of 2.0 mg. Iproniazid - Phosphate Per kg. Body Weight................... 5>1 30 Amounts of Iproniazid Found in 21+ Hour Urine Specimens Obtained From Two Rabbits That Had Received Daily Oral Doses of 2.6 mg. Iproniazid Phosrhate Per kg. Body Weight...... £2 31 Concentration of Iproniazid in Serum and Urine of 6 Male Mental Patiert s Receiving 5o nig. Marsilid Phosphate (32.ip mg. iproniazid) Daily Results of Two Analysis 15? Days Apart.... 51+ 32 Recovery of Iproniazid From Gastrointestinal Tract of Rats Following the Oral Administra­ tion of 0.98 mg. Iproniazid Phosphate.......... 56 33 Weight of Iproniazid Found in Body Tissues of Rabbits Receiving 3-25 mg. Iproniazid Phosphate Per Kg. Body Weight Daily for Four Days 58 LIST OP FIOURES Figure Page 1 Structural Formula of Iproniazid 8 2 Absorbance Spectra of Iproniazid In Acidic, Basic and Neutral Solutions (2£ Gramma per ml.).............................................. 16 viii INTRODUCTION Investigators ore presently directing a great deal of attention to mood changing drugs. However, substances affecting the moods have been known since the beginning of recorded history. The production of alcoholic beverages dates back to early Biblical days, for Noah Is reported to have made wine and to have consumed it in excess. The pioneer wine manu­ facturers probably had no idea of any therapeutic effects to be derived from their product but they must have noticed some of the effects produced when varying quantities were inbibed. The ancient Chinese who obtained alcohol by distilling fer­ mented rice observed the mental depression caused by alcohol. Thrill seekers, certain authors searching for new Ideas and persons discontented with their normal lives are among the many who consumed cannabis, cocaine or morphine for the state of euphoria experienced. This practice has been going on in some of the Oriental countries for many centuries. The modem gaseous anesthetics produce a state of ex­ citement before the patient sinks into a stupor. This was 1 2 particularly noted in the case of nitrous oxide, commonly known as "laughing gas." In the early part of the Nineteenth Century, bromides were introduced for the treatment of epilepsy, since they served to reduce the number and Intensity of the attacks. During World War II, bromides were used to quiet animals, particularily dogs, In areas subjected to frequent bombing attacks. After the Introduction of the bromides, a host of or­ ganic compounds were Introduced for use as hypnotics and soporifics. The best known members of the group include such drugs as the barbiturates, chloral hydrate, and paral­ dehyde. All of these compounds are mental depressants. The advent of the tranquilizing agents, such as mepro­ bamate, chloropromazine, and the Rauwolfia alkaloids brought about a great improvement in the treatment of mental diseases. These drugs are also very beneficial in the treatment of many anxiety states. Before the Introduction of insulin shock therapy, psychiatrists had to rely