Labetalol Hydrochloride For Transdermal Application In Pregnancy

Published Date: 02 Nov 2017

Labetalol hydrochloride is an antihypertensive agent licensed for use during pregnancy. It is licensed for use in the United Kingdom as an intravenous infusion or injection and as a tablet. Its physiological action is to competitively block the adrenergic stimulation of β2-receptors within the bronchial and vascular smooth muscle, the β1-receptors within the myocardium and the α1-receptors within the vascular smooth muscle. As a result, peripheral resistance is reduced with little effect on cardiac output.

Aim and Objectives

The aim of this research project was to conduct a pre-formulation study for a TDD system of labetalol hydrochloride for use during and after pregnancy. The hope is that this would reduce the effects of first pass metabolism and also decrease the adverse effects experienced by the baby during neonatal development and after delivery.

Method

Critical micelle concentration of SDS was estimated as 5.74mM using a curve of average conductivity and concentration data. Labetalol hydrochloride was formulated in a supersaturated solution with DMSO and an interaction was noted when SDS was incorporated. Conductivity measurements at 450nm were used to exclude the interaction between DMSO and SDS but confirmed an interaction between SDS and labetalol hydrochloride.

Results

Use of DMSO as a penetration enhancer allowed solubility of labetalol hydrochloride to increase by 1000 fold and confirmed that it could safely be used to enhance efficacy of a transdermal dose form of labetalol.

Conclusion

Chemical characteristics of labetalol hydrochloride allow for it to be a candidate for transdermal delivery. Use of dimethyl sulfoxide as a penetration enhancer in transdermal preparation allows the use of smaller concentrations of labetalol than oral formulations currently licensed for hypertension in pregnancy. Interactions of SDS and universal buffer with labetalol prevent them from use in future transdermal formulation studies. Further investigation of alternative surfactants is required in order to enhance drug solubility and delivery the intended daily dose of labetalol hydrochloride to the site of action and confer the desired therapeutic effect.

Novelty

There is an unmet clinical need for a safe antihypertensive alternative dosage form to intravenous or oral labetalol for use during pregnancy. It is important to determine a formulation that has the bioavailability to achieve sustained steady state kinetics in the required therapeutic range with minimal drug crossing through the placental barrier.

Thus far, existing studies on potential TDD systems have utilised aggressive industrial solvents unsafe for human (ex.: human skin). There is a need to determine a solvent that can serve as a penetration enhancer driving the intended dose of labetalol through the skin.

INTRODUCTION

More than four million women develop pre-eclampsia and approximately 100,000 women experience eclamptic convulsions globally, with over 90% of these cases occurring in developed countries (1). If a pregnant woman is diagnosed with pre-eclampsia, the only cure is delivery of the baby and if the diagnosis is made early in pregnancy, when the baby needs more time to mature, the mother and baby could be at increased risk if delivery is attempted (2). This makes the prevention of pre-eclampsia and the control of chronic hypertension essential to ensuring a safe and healthy pregnancy.

Hypertension during pregnancy

Hypertension is high blood pressure which can affect 1 in 10 women in the UK during pregnancy (3){Davies NM, 2003 #40}{Choices, 2011 #1}. Types of hypertension include chronic, gestational, pre-eclampsia and eclampsia.

1.1.1 Chronic hypertension

Also known as pre-existing hypertension, chronic hypertension affects 3-5% of pregnancies in the UK. It is defined as primary or secondary hypertension that presents before 20 weeks of pregnancy and continues after childbirth (4). Women with high blood pressure before week 20 of pregnancy are said to have chronic hypertension because blood pressure usually falls during the first and second trimesters (4).

1.1.2 Gestational hypertension and pre-eclampsia

Gestational hypertension and pre-eclampsia are the two types of pregnancy-induced hypertension that affect 12% of pregnancies in the UK (4). Gestational hypertension is newly diagnosed hypertension (usually stress-induced ) that presents after 20 weeks of pregnancy without significant proteinuria (protein in the urine)(2). It is more common in the second half of the pregnancy and it affects 6-7% of pregnancies in the UK (4).

Pre-eclampsia on the other hand, is defined as a disorder of high blood pressure coupled with proteinuria (5) during the second half of pregnancy in a woman who otherwise had normal blood pressure (2). It affects 5-6% of pregnancies and can have detrimental effects on both the mother and the baby (4). If un-managed, it can progress to eclampsia (5).

Women with pre-existing hypertension can also suffer signs and symptoms of pre-eclampsia. This phenomenon is known as superimposed pre-eclampsia and it affects 25% of women with chronic hypertension and this alone greatly increases the risk to the mother and the foetus (4).

Eclampsia

Eclampsia is defined as seizures that a pregnant woman suffers without a prior history of seizures or any illness affecting the brain. It is commonly a result of untreated hypertension (4).

Diagnosis and evaluations

There are no specific diagnostic and evaluation techniques for hypertension because hypertension itself is a diagnostic sign. However, other signs such as convulsions may be seen in cases of eclampsia. The determination of how severe hypertension in pregnancy is depends on the blood pressure value and the presence of proteinuria as well as other signs such as abdominal pain or malaise (4).

1.2.1 Blood pressure measurements

Mild hypertension is classified as blood pressure of 140/90 to 149/99 mmHg, moderate hypertension as 150/100 to 159/109 mmHg and severe hypertension as 160/110 mmHg or higher (3). Patients presenting with severe hypertension are normally admitted to hospital until their blood pressure decreases to at least 159/109 mmHg or lower (3). As such, prevention and management of hypertension is of concern to the NHS as it results in occupation of hospital beds that would otherwise be utilised by patients suffering from more severe ailments.

Guidelines suggest that patients suffering from mild hypertension have blood pressure readings taken not more than once a week, while moderately hypertensive patients have readings taken at least twice a week and severely hypertensive patients at least four times a week (3).

1.2.2 Proteinuria measurements

Proteinuria is defined as the presence of greater than 150 mg of urinary protein excretion per day (6). Causes of benign proteinuria (which is not associated with morbidity or mortality) include dehydration, emotional stress, fever, heat injury and most acute illnesses (6). Dipstick analysis is commonly used as a semi-quantitative measure of proteinuria (6). The results (an average of three dipstick readings) are quantified as negative (less than 10 mg /dL), trace (10-20 mg/dL), 1+ (30 mg/dL), 2+ (100 mg/dL), 3+ (300 mg/dL) and 4+ (greater than 2,000 mg/dL) (6). The National Institute of Clinical Excellence (NICE) have outlined in their guidelines assessment of proteinuria in hypertensive pregnancies. They suggest the use of automated reagent-strip reading devices to detect protein in urine in a hospital setting (3). If a result of 1+ or more is read, a 24 hour urine collection or spot urinary protein: creatinine ratio can be used to quantify the amount of proteinuria present (3). A diagnosis of significant proteinuria can be made if the protein:creatinine ratio is greater than 30 g/mmol or a 24-hour urine collection shows a result of more than 3+ (3). Patients who are mildly and moderately hypertensive should be tested for proteinuria at each doctor’s visit using either the urinary protein:creatinine ratio or an automated reagent strip reading device, while severely hypertensive patients must have the same readings taken daily (3).

1.2.3 Blood tests

Blood tests for mildly hypertensive patients should only be conducted during routine antenatal care (3). Moderately hypertensive patients should have their kidney function tested using a full blood count used to monitor transaminases, electrolytes and bilirubin (3). Unless a proteinuria diagnosis is made, additional blood tests are not recommended. In severely hypertensive patients, the same tests should be conducted at presentation of severe blood pressure and monitored weekly in addition (3).

1.3 Impact of pre-eclampsia on the National Health Service

Control of hypertension in pregnancy is of importance to the NHS. This is because 5% of stillbirths occur in women with pre-eclampsia (3) and studies show that 0.4% (1 in 240) women will give birth before their 34th week of pregnancy as a result of pre-eclampsia, and 8%-10% of all preterm births are a result of hypertensive disorders where half of women with severe pre-eclampsia give birth (3). Monitoring for pre-eclampsia and eclampsia is strongly advised during pregnancy and pregnant women have blood pressure and protein urea levels measured routinely.

Clinical guidelines recommend that diagnosis and management of hypertension during pregnancy should be conducted during the antenatal, intrapartum and postnatal periods (3). Physicians should take into consideration the summary of product characteristics (SPCs) for each medicine in order to tailor anti-hypertensive treatment to each individual patient, paying attention to contraindications, cautions and interactions (3).

Lifestyle and treatment advice for pre-eclamptic women

Pregnant women with chronic hypertension are advised to reduce or substitute sodium in their diets so as to reduce their blood pressure (7). Women with organ damage such as kidney disease secondary to chronic hypertension should have a target blood pressure of less than 140/90 mmHg (7). Those with secondary chronic hypertension should be referred to a specialist of hypertensive disorders (7). Pregnant women with chronic hypertension should only be prescribed treatment based on pre-existing treatments, teratogenicity risk factors and side-effect profiles (7).

Antenatal consultations, timing of birth and postnatal investigations

Based on the needs of the mother and the baby, antenatal consultations can be scheduled for pregnant women with chronic hypertension. If the blood pressure of the woman is lower than 160/110 mmHg and she has not been taking an antihypertensive prior to 37 weeks, birth should not be offered and the indications for birth must be agreed upon by the woman and a senior obstetrician (7). Women with refractory severe chronic hypertension should only be offered birth after a course of corticosteroids (if required) has been completed (7). In post-natal care, the target blood pressure should be below 140/90 mmHg. Women with chronic hypertension should have their blood pressure measured daily for the first two days following birth, at least once between the third and fifth day after birth, and as clinically required if antihypertensive treatment is changed after birth (7).

Guidelines on the management of pregnancy-induced hypertension

1.6.1 Treatment in secondary care

Women in hospital settings who have suffered an eclamptic fit should be treated with intravenous magnesium sulphate. The same treatment can also be offered to women in a hospital setting with severe pre-eclampsia due to give birth within 24 hours (7). Antihypertensive medicines that are licensed for use in women with severe hypertension during pregnancy or after birth include labetalol (oral or intravenous), intravenous hydralazine, and oral nifedipine with the target blood pressure below 150 mmHg and diastolic pressure 80-100 mmHg (7). In women with pre-eclampsia with birth due within 7 days, corticosteroid such as betamethasone intramuscularly is given for foetal lung maturation. In breastfeeding, antihypertensive drugs are not known to have adverse effects on babies receiving breast milk. Examples of such include labetalol, nifedipine, enalapril, captopril, atenolol and metoprolol (7). On the other hand, there is insufficient evidence on the safety in babies receiving breast milk from a mother taking angiotensin receptor blockers, amlodipine, enalapril and captopril (7).

Treatment options should be discussed openly with the patient and all information should be made equally available to patients with efforts made to meet their additional needs especially if they have language barriers, learning, sensory or physical disabilities (3).

1.6.2 NICE guidelines for treatment of pre-eclampsia

Guidelines from NICE suggest that women who are at high risk of pre-eclampsia should take 75 mg of aspirin daily from their twelfth week of pregnancy until the day of birth. Women considered at risk are those with any one of: hypertension during a prior pregnancy, chronic kidney disease, auto immune disease such as systemic lupus erythematosus or anti-phospholipid syndrome, type 1 or 2 diabetes, or chronic hypertension (3).

1.6.3 NICE guidelines for treatment of chronic hypertension

Women who suffer from chronic hypertension and ACE inhibitors and angiotensin II receptor blockers (ARBs) should be advised of an increased risk of congenital abnormalities if medicines in these classes are taken during the pregnancy (3), and urged to discuss other anti-hypertensive treatment that they plan to use during pregnancy (3). During treatment, the degree of hypertension is determined based on the blood pressure levels.

1.6.4 Contraindications in medicinal therapy

All of the medicinal therapies licensed for use in pregnancy and postnatal obstetric practice come with warnings against use if the risk of toxicity outweighs the benefits. Since treatment during pregnancy simultaneously affects two individuals (mother and baby), the chances of toxicity or teratogenicity are increased. Beta-blockers are most commonly used and they have various contraindications. Atenolol is noted to have increased risks in its use during the first and second trimester (such as intra-uterine growth restriction, neonatal hypoglycaemia, and bradycardia(8), and labetalol is recommended for use only in the first trimester of pregnancy and in breastfeeding (7, 8). ACE inhibitors are also contraindicated in various terms of the pregnancy. Captopril and enalapril are contraindicated in the second and third trimesters and are not recommended in the first trimester (7).

1.6.5 Toxicity associated with pregnancy

Foetal growth impairment is a concern when treating hypertension in pregnancy. The placental blood barrier is relatively ‘leaky’, and allows diffusion of xenobiotics into the amniotic fluid (9). The main mode of transport across the placental blood barrier is through passive diffusion. It is favourable to lipophilic agents and placental blood flow is the rate-limiting step (10). Passive diffusion across the placental barrier depends on a concentration gradient between maternal and foetal blood, pH of the maternal and foetal blood and protein binding (11). Foetal exposure to xenobiotics is dependent upon the combination of transporters on the placental-blood barrier, and their substrate specificity (12). 

1.6.6 Pharmacokinetic changes in pregnancy

During pregnancy, changes in absorption, distribution and clearance of medicines, are significantly noted in the third trimester (10). Absorption is decreased as a result of raised progesterone, reduction in gastric emptying and decreased motility in the small intestine (10). This increases the time to peak concentration (Tmax) and decreases the peak concentration (Cmax). Absorption of orally administered medicines is also affected by the nausea and vomiting related to pregnancy which can be remedied by administering an evening dose when nausea is lessened (10). Distribution is affected in pregnancy when total body water increases (up to 8 litres), resulting in an increase in the volume of distribution (Vd) and a decreasing in the Cmax. Consequently, clearance of medicines significantly changes as renal blood flow increases by 60-80% and glomerular filtration rate increases by 50%, resulting in enhanced elimination of medicines that are normally excreted in unchanged form (10). Hepatic blood flow is also increased in medicines with high hepatic extraction ratios.

1.7 Labetalol

1.7.1 Chemical structure and properties

Labetalol, whose structure is shown in Figure 1, is an anti-hypertensive that works as a mixed adrenergic antagonist. It exerts its effects on α- and β-adrenocepters. It has a molecular formula of C19 H24 N2O3 and a molecular weight of 328.4.

Figure

Figure 1. Labetalol chemical structure

Labetalol hydrochloride chemically fits the characteristics of a suitable candidate for transdermal patch formulation. It has a molecular weight preferably less than 500 Daltons (364.9 Daltons for labetalol), a melting point that is less than 200 oC (188 oC for labetalol), a half-life of less than 6-8 hours (3-6 hours for labetalol). Clinically and therapeutically, labetalol also qualifies for transdermal formulation because first-pass metabolism will be circumvented. Continuous zero-order drug administration, increased bioavailability, and easier dosage regimen will improve therapeutic outcome and increase compliance. Additionally, terminating therapy by simply removing the patch if side effects are experienced is an added advantage of this formulation (13). In depth discussion and application of TDD formulation will be discussed further in this project.

1.7.2 Physiochemical properties of labetalol

Labetalol hydrochloride has two chiral carbons and four stereoisomers as shown in Figure 2 (14). The SS and RS-isomers are inactive while the SR-isomer form causes majority of the α1-adrenergic blockade and the RR-isomer (dilavalol) exhibits mixed β and minimal α1-adrenergic-blockade (15).

Figure 2. Stereoisomers of the Labetalol chemical structure (chiral carbons (*)) (14)

Oral labetalol is classified as a Class I agent (highly soluble, highly permeable and rapid dissolution) in the Biopharmaceutics Classification System (BCS) see Figure 3.

Figure 3. Biopharmaceutics classification of oral labetalol hydrochloride (16, 17)

Due to extensive first-pass metabolism, only about 25% of the oral dose of labetalol is bioavailable (17). Class 1 agents are considered to have rapid and complete absorption sometimes greater than 90% (17) and their rate of dissolution is limited by in vivo absorption (18). The rate-limiting step to absorption of class 1 medicines such as labetalol is gastric emptying (17). Captopril another class 1 antihypertensive agent which has the same amphiphilic properties as labetalol while atenolol has more hydrophilic properties and is a class 3 agent whose permeability is the rate determining factor (18).

1.7.3 Stability

Tablets of labetalol hydrochloride should be stored at 2-30 °C in secure containers, and if packaged as unit-doses, they should be protected from moisture (19). The intravenous injections should also be stored at 2-30 °C, safeguarded against freezing and kept away from light (19).

1.7.4 Preparations

Labetalol hydrochloride is available in both branded and generic (non-proprietary) names and is obtainable from more than one manufacturer or distributor as shown in Table 1 (8).

Table . Preparations of labetalol hydrocholide licensed and marketed in the UK (8).

Routes

Dosage Forms

Strengths

Brand Names

Manufacturer

Oral

Tablets, film-coated

50 mg

Trandate® 

PharSafer

100 mg

Labetalol Hydrochloride Tablets

 

 

 

 

Trandate® (scored)

PharSafer

 

 

200 mg

Labetalol Hydrochloride Tablets

 

 

 

 

Trandate® (scored)

PharSafer

 

 

400 mg

Labetalol Hydrochloride Tablets

 

 

 

 

Trandate® (scored)

PharSafer

Parenteral

Injection, for i.v. use

5 mg/mL

Labetalol Hydrochloride Injection

 

1.7.5 Pharmacokinetics of labetalol

1.7.5.1 Absorption

Rapid absorption (~90-100%) of labetalol occurs in the gastrointestinal tract following oral administration (20).

1.7.5.2 Bioavailability

Variation between 10-80% is observed in bioavailability with a mean of 30%, and a plasma half-life of 3-6 hours (21).

1.7.5.3 Distribution

The volume of distribution is 3-10 L/kg with an average volume of distribution of 7L/kg. The volume of distribution increases in pregnant women, and those with impaired liver function (22). After intravenous administration, labetalol is rapidly and widely distributed into the extravascular space (22). About 25% of the medicine reaches the systemic circulation in unchanged form in fasted adults since food decreases first-pass metabolism (22). Although labetalol crosses the placental-blood barrier and small amounts of unchanged medicine have been noted to distribute in the foetal uveal tract and in breast milk, NICE guidelines suggest that women should be advised that labetalol has no known adverse effects on infants who are breastfeeding (3).

1.7.5.4 Metabolism

Labetalol hydrochloride undergoes extensive hepatic first-pass metabolism following oral administration and 50% protein binding (23).

1.7.5.5 Elimination

Concentrations of labetalol in the blood plasma decrease in a biphasic or even triphasic manner (21). In 24 hours, only about 5% of the medicine is excreted unchanged, but majority of it is excreted in form of O-alkylglucuronide (major metabolite), with O-phenylglucuronide and N-glucuronide in lesser amounts (20) and a plasma clearance of 10-40 mL/min/kg with an average of 25 mL/min/kg is observed (21). Most of the dose is metabolised in the liver and metabolites are excreted within 24 hours as glucuronide conjugates through the urine (60% of the dose). Within 4 days, about 30% of the dose is excreted through the faecal route (20). Less than 1% is eliminated by peritoneal dialysis or haemodialysis (21).

1.8 Treatment of hypertension in pregnancy

Before anti-hypertensive treatment can commence for women with gestational hypertension, there are some risk factors that must be considered such as family history of pre-eclampsia, multiple pregnancy, body mass index exceeding 35 kg/m2, patient’s age (over 40 years), pre-existing vascular or kidney disease as well as pregnancy interval period greater than 10 years (3).

1.9 Use of labetalol hydrochloride

Labetalol is legally classified as a prescription only medicine (POM) and is licensed for treatment of hypertension in pregnancy (8). It can also be used during breastfeeding because the amount of medicine present in breast milk is not enough to jeopardise the growth of the infant (8). There is no evidence thus far that associates the use of labetalol with an increased risk of foetal deformity. Although use of beta blockers during the first and second trimesters of pregnancy have been associated with intrauterine growth retardation and low birth weight, these effects are uncommon when labetalol is used (8). Conversely, neonatal beta adrenoceptor blockade leading to hypoglycaemia, neonatal bradycardia, respiratory distress, apnoea and hypotension may result when labetalol is used near term, although these symptoms are reported to be mild and tend to resolve within 48 hours of delivery (24). To reduce the likelihood of this occurring, treatment with labetalol should be discontinued 24-48 hours before delivery (25).

Women treated with labetalol before pregnancy are advised to continue taking it during pregnancy as well in order to minimise maternal and foetal risks of hypertension (3). Since labetalol is the safest antihypertensive treatment, NICE suggests that alternatives such as methyldopa and nifedipine only be used after considering side-effects to the woman, foetus and new born baby (3).

1.9.1 Oral dosage form

Oral labetalol is recommended by the NICE as first line therapy for treatment of moderate to severe hypertension in pregnancy (3). Severe hypertension (where blood pressure is ≥180/110 mmHg) without acute target-organ damage is described as hypertensive urgency (8). The aim in controlling hypertensive urgency is to gradually decrease the blood pressure over 24-48 hours using an oral dose of labetalol or other antihypertensive (8).

The licensed oral dose for treatment of hypertension in pregnancy is 100 mg twice daily (8). The dose can be increased at weekly intervals by 100-200 mg per day until sufficient reduction in blood pressure is noted or side effects prevent further increase in dose (8). During the second and third trimesters or due to increasing severity of hypertension, the dose may be titrated so that 100-400 mg of labetalol can be taken three times daily (3). The maximum recommended dose is 2400 mg daily (8).

1.9.2 Intravenous dosage form

Labetalol by intravenous injection is licensed for use in hypertensive emergency (8). A hypertensive emergency can be described as severe hypertension with acute damage to the target organs where rapid treatment with intravenous antihypertensive medicine is required (8). A dose of 50 mg over at least 1 minute is advised, and can be repeated after 5 minutes if required for no more than a maximum total dose of 200 mg (8). Within the first few minutes to 2 hours of treatment with intravenous therapy, blood pressure should be decreased by 20-25% (8).

1.9.3 Side-effects

Side effects including headache and tiredness are most commonly reported (8). Dizziness and postural hypotension are experienced at higher doses and rarely liver damage may occur (8). If liver damage is suspected, treatment should be stopped and liver function tests should be carried out (8).

1.9.4 Contra-indications

Labetalol is contraindicated in patients who have hypotension, bradycardia (heart rate less than 45-50 beats per minute), second or third degree heart block, metabolic acidosis, hypersensitivity to labetalol, cardiogenic shock, untreated pheochromocytoma, history of wheezing or asthma, uncontrolled heart failure, severe peripheral circulatory disturbances, sick sinus syndrome (including sino-atrial block) and Prinzmetal’s angina (26).

1.9.5 Overdose

Therapeutic overdose with labetalol (or another beta blocker) can result in dizziness and light-headedness and perhaps syncope due to bradycardia and hypotension (8). In the case of an overdose or hypersensitivity (27), the patient should be closely supervised and treated in secondary care in an intensive care unit (ICU).

Activated charcoal and laxatives can be used to reduce the absorption of the medicine in the gastro-intestinal tract (27). Additionally, gastric lavage can also be use and artificial respiration may be required (27). Excessive bradycardia can be treated with an intravenous injection dose of atropine sulphate 0.6-2.4 mg in divided doses of 600 μg (8). Haemodialysis only removes less than 1% of labetalol from the circulation (27).

1.10 Transdermal dosage form

Transdermal formulations are beneficial because they avoid first pass metabolism, they provide a continuous delivery of treatment, and compliance is increased because fewer dosage units are required to provide the desired clinical effect (28).

1.10.1 Transdermal drug delivery (TDD) systems

Several indications have been successfully treated using TDD. Examples include motion sickness (hyoscine), cardiovascular disease (clonidine and nitroglycerin), chronic pain (fentanyl), smoking cessation (nicotine), hormone replacement- oestradiol (alone or in combination with norethisterone or levonorgesterel) (29). Although only a few medicines are currently marketed as TDD systems, there are many more that are undergoing formulation and clinical development for indications such as Parkinson’s disease, Alzheimer’s disease, anxiety, depression, skin cancer, and many more (29). The global revenue that transdermal products yield is estimated to be 3 billion USD between USA (56%), Europe (32%) and Japan (12%) with revenue growth expected to increase (29).

When the medicine in a TDD system comes in contact with the skin, it can be absorbed into the skin surface by one of three pathways: the sweat ducts, hair follicles and sebaceous glands or directly across the stratum corneum layer of the epidermis (Figure 4.) (29).

Figure 1 :Simplified representation of skin showing routes of penetration: 1. through the sweat ducts; 2. directly across the stratum corneum; 3. via the hair follicles

Figure 4. Possible pathways for the drug to penetrate the skin: 1. through the sweat glands 2. through intact stratum corneum, and 3. through hair follicles associated with sebaceous glands (29).

The use of TDD systems on a wider range of treatments is limited by the inability of drug molecules to penetrate the outer most layer of the skin known as the stratum corneum (29). The daily dose that can be administered through a transdermal patch is 5-10 mg and that too limits potential candidates even further to those that are potent enough to be administered at this low dose (29). (29). The concentration of parent drug molecules that are able to permeate through the skin obey Fick’s first law (which relates the concentration gradient to the flux of atoms passing through unit area in unit time) denoted by Equation 1 as follows:

(1)

where J is the steady-state flux which is correlated to the diffusion coefficient D of the drug in the stratum corneum layer over the partition coefficient between the vehicle and the stratum corneum, h is the membrane thickness, and C0 is the constant applied drug concentration (29).

Physical and chemical enhancement techniques can be applied to increase penetration of drug molecules across human skin (29). Examples of physical techniques include jet-injectors, electroporation, micro needles, iontophoresis and phonophoresis (29). This research project however focused more on the use of a chemical to enhance penetration and increase the drug concentration and solubility.

Surfactants

In order to formulate a drug into a TDD system, the drug must be solubilised in an appropriate medium (30). Surfactants are used to increase solubility of the drug to ensure sufficient penetration through the layers of the skin. There are various natural surfactants present in the body such as cholesterol (and its esters), bile salts and lecithin (30). However, these are only effective if the drug is being administered orally and will enter the gastro-intestinal tract. For the purpose of this research study, SDS was considered to increase the solubility of labetalol hydrochloride.

1.11 DMSO as a chemical penetration enhancer

Dimethyl sulphoxide (DMSO) is one of the most commonly researched penetration enhancers and has been referred to as the ‘universal solvent’ (31) because of its ability to disrupt the multiple lipid membranes between skin layers and the cells to make intracellular drug penetration more effective. Additionally, DMSO has a higher boiling point than water of 189ºC (32) indicating that DMSO does not evaporate when in contact with human skin or at body temperature, enabling the drug to stay in solution and penetrate into the body targeting the site of action.

It is hydroscopic, colourless, odourless and alone it has been used topically to treat systemic inflammation (31). Despite its success as a penetration enhancer, DMSO in high concentrations (greater than 60%) denatures skin proteins (31). DMSO can also cause erythema (redness of the skin), burning sensation, scaling, stinging and contact urticarial (31).

1.12 Penetration of labetalol in skin

A previous study of transdermal labetalol hydrochloride has been conducted on albino rat skin using penetration enhancers (33). Various penetration enhancers were used in order to alter permeation. Examples of enhancers used were turpentine oil, dimethyl formamide, menthol, DMSO, pine oil and 2-pyrollidone in 5% v/v concentration. Results from this study confirmed that labetalol was a suitable candidate for transdermal formulation using 5-10% DMSO as a penetration enhancer.

2. HYPOTHESIS, AIMS AND OBJECTIVES

2.1 Hypothesis

Based on results from a previous study, administering labetalol hydrochloride through the transdermal route will yield zero-order kinetics. The mother could also benefit from longer anti-hypertensive control while the neonate will be exposed to lower concentrations of the drug because there will be less drug present to pass through the placental blood barrier.

2.2 Aims

The aim within this project is to carry out a pre-formulation research study of transdermal dosage of labetalol hydrochloride for potential use in pregnancy-induced hypertension by considering surfactants and solvents as possible ways of increasing concentration which may also improve penetration.

2.3 Objectives

To meet the aim of this research study, the objectives were to:

Determine the absorbance of labetalol in pH 7.4 phosphate buffer in varying known concentrations

Determine the CMC value of SDS by measuring the conductivity of SDS in pH 7.4 phosphate buffer

Investigate interaction between labetalol and SDS using conductivity readings

Determine maximum solubility of labetalol for potential transdermal dosage form in a supersaturated aqueous solution from DMSO

3. MATERIALS AND METHODS

3.1 Equipment and apparatus.

Cecil CE1021 Spectrophotometer

Grant XUBA3 ultrasonic bath

Clifton cyclone whirly mixer

Mettler Toledo FE20 pH meter

Precisa 125A digital weighing balance

Jenway conductivity meter

Quartz glass cuvettes

The standard operating procedures (SOPs) for safe usage of the above apparatuses are available in section 8.4.

3.2 Chemicals

Universal buffer (pH 7.0), phosphate buffer (pH 7.4) and sodium acetate buffer (pH 4.5) were prepared in the laboratory as detailed in section 8.1.

Labetalol hydrochloride and DMSO were obtained from Sigma-Aldrich and SDS was obtained from Fisher scientific.

3.3 Cleaning cuvettes

To reduce the likelihood of poor reproducibility, inaccuracies and lack of precision, the inside and outside surfaces of the cuvettes were cleaned using a procedure detailed in section 8.4.

3.4. Calibration curve

A calibration curve is an experimental estimate of the relationship between a concentration and an experimental absorbance reading corresponding to that concentration. The aim of a calibration curve is to determine the relationship between the signals produced by a solution of unknown concentration (containing the drug sample) compared to signals produced by samples of known concentration (the standard). Labetalol hydrochloride was made up in known concentrations in both universal and phosphate buffers, and calibration curves were plotted with the expectation that a linear relationship between concentration and absorbance would be established and the data would be considered reliable.

3.4.1 Calibration curve for labetalol in universal buffer

Absorbance values of labetalol in universal buffer were obtained using the Cecil spectrophotometer with instructions from section 8.4.

Labetalol (10.5 mg) was weighed and dissolved in 100 ml of universal buffer as detailed in Section 8.16 .

The spectrophotometer was zeroed using 3 ml of the universal buffer and an absorbance reading of 0.00 was recorded.

Using another clean Pasteur pipette, a sample (enough to have the absorbance light beam pass through) of the 0.105 mg/ml solution was put into another clean cuvette and it was placed in an adjacent chamber to the blank sample. The chamber cover was closed and an absorbance of 0.51 was recorded.

A serial dilution was performed between concentrations of 0.105 mg/ml – 0.013 mg/ml (section 8.1), and repeated twice more using initial concentrations of 0.10 mg/ml and the same trend of lower than half absorbance levels was recorded.

3.4.2 Calibration curve for labetalol in phosphate buffer

Phosphate only buffer (pH 7.4) was prepared using 10 mM sodium dihydrogen orthophosphate and 10 mM disodium hydrogen orthophosphate.

Serial dilutions of labetalol hydrochloride in phosphate buffer were made in a similar fashion (see section 8.1).

3.5 Determination of the CMC value of SDS

Conductivity measurements of SDS solutions in phosphate buffer were performed. A stock solution of 2.31 g of SDS in 500 ml buffer was used to prepare SDS solutions of known concentrations and their conductivity was measured using a Mettler Toledo FE20 pH meter (as per instructions detailed by section 8.4). A curve of average conductivity versus SDS concentration was plotted and the curves of the upper and lower ranges above the estimated CMC value.

3.6 Supersaturated solution of labetalol

Transdermal formulations deliver the maximum dose if the drug is in a supersaturated solution because the thermodynamic activity of the drug is at its highest (29). For this study, labetalol hydrochloride was mixed with phosphate buffer but due to the high solubility of labetalol in water, a point of saturation was not reached although an excess of 91.1 mg of labetalol hydrochloride was dissolved in 20 ml of phosphate buffer. The chemical properties of labetalol were considered. Since the pKa of labetalol is 7.4 and 8.7, the use of a more acidic buffer was considered. Sodium acetate buffer pH 4.5 was prepared (see section 8.1) and used. A 20 mM stock solution of 2.88 g SDS in 500 ml 10 mM sodium acetate pH 4.5 buffer was made (with the 2.88G of SDS).

Similar to the solution in phosphate buffer, an excess of 96 mg of labetalol hydrochloride was added to 20 ml of the sodium acetate buffer and a saturated solution was not achieved.

DMSO solutions in increasing drug concentration (detailed in section 8.2), were added to SDS and mixed with phosphate pH 7.4 and acetic acid pH 4.5 buffers. Their UV absorbance was measured at 450 nm until scattering of the signal or a cloudy solution was formed indicating the formation of a precipitate and the maximum concentration of the solubilised drug that could be incorporated into a transdermal patch (as shown in section 8.2).

3.7 Analysis of the interaction of drug with SDS

A conductivity study was performed using DMSO solutions of labetalol in the presence of SDS and phosphate pH 7.4 buffer to investigate the apparent interaction. Conductivity of solutions of known SDS concentration in phosphate buffer was measured without the presence of labetalol, and in the presence of labetalol, immediately after the drug was added and a few hours after the drug was added to give the solution enough time for any notable interaction to occur (see section 8.2).

3.8 Labetalol hydrochloride in an aqueous solution

The solubility of labetalol hydrochloride in water is 117 mg/L (in 25 °C) (22). In order to achieve a solution of more than 200 mg (the recommended daily dose to treat hypertension in pregnancy) in a transdermal patch, DMSO was used as a vehicle to dissolve a higher concentration of the drug. Solubility of 240 mg of labetalol in 1 ml of DMSO was achieved, which is more than 1000 fold increase compared to the known solubility of labetalol in water (0.12 mg/ml).

3.9 Statistical analysis

Statistical assessment of all data was performed. Each measurement was performed in triplicate in order to perform statistical analysis and conclude that the data obtained was reliable and reproducible. Using Microsoft Excel, the average of the values obtained in all experiments was calculated using Equation 2 as follows:

(1)

where Σ is the sum of X, the individual data points and N is the sample size (number of data points). Using the mean of the data, the standard deviation was also calculated in order to measure the dispersion or spread of the individual values from the mean value sing Equation 3 as follows:

(2)

where  is the sum of X the data points, M is the mean of the data points, and n is the sample size (the number of data points). Sample error (SE) was calculated to show the error between the true average and the measured average as follows:

(3)

as the sample size increases the SE decreases. It is more appropriate in showing a statistically significant difference. A T-test was used to compare the means of three samples in relation to the variation in the data, which is expressed as the standard deviation of the means of the sets of data.

4. RESULTS AND DISCUSSION

4.1 Calibration curves

A calibration curve was obtained to determine the relationship between known concentrations of labetalol hydrochloride and absorbance values. At a wavelength of 300 nm, the absorbance of labetalol in phosphate buffer was measured as detailed in section 3.4.2. Three replicates were used to increase the reliability of the results and ensure that the values obtained are reproducible.

Figure 5. Calibration curve of triplicate absorbance measurements of Labetalol hydrochloride in pH 7.4 phosphate buffer at 300nm. The mean value ± SE (n=3) was plotted for each concentration of labetalol used. R2 value was found to be 0.9993 and the line of best fit was y = 6.8561x.

Absorbance increased as labetalol concentration increased. The correlation coefficient (R2) is greater than 0.99 that suggests that the linear regression line has a strong correlation with the overall trend. The high R2 value also suggests that using the equation produced from the line of best fit, absorbance values can be input into the equation to predict concentrations that can be used to generate other experimental designs.

4.2 Critical Micelle Concentration

CMC is a surfactant property that illustrates the concentration above which the formation of micelles occurs.

An anionic surfactant such as SDS is an example of a detergent with a hydrocarbon chain and a negatively charged sulfonate group.

The conductivity of SDS in phosphate buffer was used to estimate the CMC. Conductivity tests of SDS solutions in phosphate buffer were performed as detailed in section 3.5.

Figure 6. The mean conductivity (n=3) of each concentration of SDS dissolved in phosphate buffer was plotted. The line of best fit as y = 36.921x + 1563.4 and R2 = 0.9554. The upper range above CMC concentration (* * *) and the lower range below the CMC concentration (- - -) were used to produce lines of best fit that allowed the estimation of the CMC value.

As SDS concentration in phosphate buffer solution increased, so did the conductivity due to mobile SDS ions. However, there was a decrease in the slope of this increase as the concentration increased past the CMC value, indicative of when the surfactant went into the micelles, reducing the mobile ions. General formulae of conductivity as a function of concentration were obtained for both lines and the CMC (the point at which the two lines intersect) was estimated as follows.

4.3 Absorbance Trends

Saturated aqueous drug solutions are used in transdermal delivery of treatment doses across the skin barrier into circulation. To test for efficacy in solubility of labetalol for potential transdermal formulation, aqueous solution with DMSO was formulated. Neutral and acidic buffers were used in the absence of SDS as a negative control sample and the trend was as follows.

Figure 7. Absorbance readings of DMSO and labetalol hydrochloride in the absence of SDS. Neutral buffer pH 7.4 () and acidic buffer pH 4.5 () show linear increase in absorbance and no interaction with labetalol hydrochloride in DMSO concentration of 0.010-0.250mM.

Into the negative control samples, small concentrations of SDS were added and absorbance was measured to ensure that the surfactant could be used in transdermal formulation with labetalol and DMSO and improve permeability of drug through the stratium corneum. An interaction was noted immediately with both buffers as detailed below (Figure 8). Turbidity was also observed as drug and surfactant interacted. At such low concentrations of labetalol hydrochloride, therapeutic outcomes would not be achieved in the presence of SDS since most of the drug would interact with the surfactant and not reach the site of action.

Figure 8. Absorbance readings of DMSO and labetalol hydrochloride in the presence of SDS. Neutral buffer pH 7.4 () and acidic buffer pH 4.5 () show linear increase in absorbance and an interaction with labetalol hydrochloride in DMSO concentration of 0.0010-0.0350mM.

4.3 Conductivity Trends

To further analyse the noted interaction between labetalol and SDS, conductivity readings were taken in the presence of neutral phosphate buffer pH 7.4. Readings were taken at different intervals of when the drug was added to solution. The negative control samples produced a linear curve (Figure 9).

Figure 9. Conductivity of SDS solution and phosphate pH 7.4 buffer in the absence of Labetalol hydrochloride as a negative control. Linear increase in conductivity is noted and a shift in CMC compared to Figure 6 as upper range above CMC concentration (* * *) and the lower range below the CMC concentration (- - -) intersect at a point slightly more to the left of 5.74 mM.

A solution of labetalol hydrochloride with DMSO was added to the SDS and buffer solutions. Conductivity was measured immediately thereafter and the CMC was seen to shift as follows.

Figure 10. Conductivity of SDS solution and phosphate pH 7.4 buffer immediately after addition of Labetalol hydrochloride. Conductivity increased and the CMC shifts slightly to the right.

Samples containing labetalol were kept in room temperature for a few hours allowing any further interaction to occur and for better conclusions to be made regarding the interaction between the surfactant and labetalol. Approximately five hours later, conductivity was measured and the conductivity decreased (Figure 11).

Figure 11. Conductivity of SDS solution and phosphate pH 7.4 buffer approximately 5 hours after addition of Labetalol hydrochloride.

Statistical analysis of all three curves yielded the results presented below.

Conductivity in absence of drug (µS)

Conductivity immediately after addition of drug (µS)

Conductivity ~5 hours after addition of drug (µS)

Mean

1620

1741

1714

Standard Deviation

150.9

217.9

205.6

P-value between conductivity taken without presence of the drug and immediately after the addition of drug = 4.53E-08

P-value between conductivity taken without presence of the drug and ~5hrs after the addition of drug = 0.00420

P-value between conductivity taken immediate addition of drug and ~5hrs after addition of drug = 4.38E-07

A one tail T-test between the conductivity readings taken without the drug and readings taken approximately 5 hours after the drug was added showed statistical significance (p< 0.05) and therefore there was a considerable shift in CMC values and a confirmed interaction between labetalol hydrochloride and the SDS surfactant. All of the p-values are significant, i.e. less than 0.05; indeed, the first and third are even more significant, being less than 0.001.

5. FINAL CONCLUSIONS

In conclusion, chemical characteristics of labetalol showed that it has the desired characteristics to penetrate the skin. It has a molecular weight of less than 500 Daltons, melting point less than 200 o C and half-life of less than 6-8 hours. Compared to oral dosage form, a transdermal delivery of labetalol would avoid first pass metabolism and less amount of the drug would be used to achieve the daily dose. Consequently, multiple dosing would not be required because depending on the type of patch used, one application could last for hours or even days. This would allow for steady state kinetics compared to the peaks and troughs seen the in the profile of oral administration and ensure that hypertension in pregnancy is controlled with minimal risk of developing pre-eclampsia or eclampsia. This would increase compliance and reduce the chances of blood pressure and related complications such as stroke, heart attack or cardiovascular disease. Transdermal delivery is also a non-invasive form unlike oral and intravenous in that if side effects are experienced, dosing can be stopped by removal of the patch. This allows for better monitoring and control of both therapeutic and side effects of the drug.

This research project also reported that smaller concentrations of the drug were used when in solution with DMSO compared to aqueous media alone. This was evidence that smaller amounts than those used in oral formulation would be required to deliver the intended daily dose, thus providing an economic, and compliance. In a previous study, the use of more permeability enhancers such as DMSO, DMF, turpentine oil, pine oil, menthol and 2-pyrollidone, were tested (33). Results showed an increase of the permeability coefficient of labetalol by close to 17% when 5% DMSO was used (33). DMSO of concentration 5-10% was also stated to be the most suitable permeability enhancer for use with labetalol in transdermal drug delivery systems (33) as the permeability coefficient of labetalol was seen to increase as the concentration of DMSO increased from 5 to 7.5 to 10% (33).

6. RECOMMENDATIONS

Using the data obtained in this pre-formulation study, further work on the identification of a surfactant other than SDS, which does not interact with the drug, could prove to be beneficial. Surfactants with low toxicity have been proven to improve the flux of drugs through biological membranes where they adsorb at interfaces and interact with membranes, and result in increased penetration of the drug through the layers of the skin (34).

In addition to only one surfactant being tested, this pre-formulation study also used one solvent (DMSO) as a penetration enhancer. Consideration of other enhancers in future experiments could offer safer alternatives that are non-allergenic when in contact with skin and effective at the same time.

Various studies that consider different vehicles have been published and they have been shown to combinations of these enhancers to achieve maximum permeability (2). Similar experiments can be done using labetalol and the change permeability coefficient across the skin can be studied.

For clinical purposes, the rate of penetration of a dose of labetalol through the skin could be further explored in order to determine the daily dose and maximum daily dose that can be formulated in each patch. Doing so would determine the extent to which compliance can be affected by using a patch versus the oral or intravenous doses already licensed for use. Skin permeation experiments could be conducted in vitro using different concentrations of DMSO and changes in the permeability coefficient would be used to determine the suitability of labetalol hydrochloride to be delivered cutaneously.

The rate of drug release from different delivery systems can be studied to determine the most effective one in delivery of the intended dose of labetalol hydrochloride through the skin. Some TDD system designs dissolve the drug in a liquid (as pre-formulated in this study) or in a gel-based reservoir (34). The type of patch design determines the frequency of application and this would be of clinical relevance to labetalol formulation because it would dictate the level of compliance that can be expected as well as the location on the body upon which the patch can be applied.

Overall, this formulation of labetalol hydrochloride is promising in its clinical value to the patient and economic value to the manufacturer.